CN105378070A

CN105378070A - Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells

Info

Publication number: CN105378070A
Application number: CN201480022584.0A
Authority: CN
Inventors: R·P·兰扎; 卢世江; 汪维
Original assignee: Advanced Cell Technology Inc
Current assignee: Astellas Institute for Regenerative Medicine
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2016-03-02
Also published as: IL294897A; IL294897B2; EP2970903A1; IL241177A0; WO2014145108A1; JP2016515382A; EP2970903B1; EP4292600A2; CA2906815A1; KR102263956B1; JP2022023092A; KR20160002779A; IL276173A; JP2024053564A; DK2970903T3; AU2014233403A1; AU2014233403B2; EP2970903A4; US20220347228A1; AU2022201986A1

Abstract

Methods are provided for the production of photoreceptor cells and photoreceptor progenitor cells from pluripotent stem cells. Additionally provided are compositions of photoreceptor cells and photoreceptor cells, as well as methods for the therapeutic use thereof. Exemplary methods may produce substantially pure cultures of photoreceptor cells and/or photoreceptor cells.

Description

The sight sensor produced by multipotential stem cell and sight sensor progenitor cell

Related application

According to 35U.S.C. § 119 (e), this application claims the U.S. Provisional Application Ser No.61/793 being entitled as " PHOTORECEPTORSANDPHOTORECEPTORPROGENITORSPRODUCEDFROMPLU RIPOTENTSTEMCELLS " submitted on March 15th, 2013, the rights and interests of 168, the full content of this application is incorporated herein by reference.

Background technology

Retinal diseases causes blind owing to losing the neuronal cell after mitotic division usually.Retinal diseases comprises rod photoreceptor cell or Progressive cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis and Stargardt are sick.In most retinal degeneration, mainly on outer nuclear layer, loss cell occurs, wherein said outer nuclear layer comprises retinal rod and cone photosensory cell.When after losing mitotic division when neuronal cell population, need the new cell replacement photosensory cell of external source.

The potential alternative source of photoreceptor cell comprises stem cell.Early stage research employs the heterogeneous population of mouse cell, mouse stem cells or retinal progenitor cells as the possible cell derived for substituting the sight sensor lost.These early stage researchs describe the sight sensor precursor cell (Maclarenetal.Nature444 (9): 203-207,2006) transplanted and obtained by the retina of the 1st day mouse after being born; By the external generation retinal precursor cells of the embryonic stem cell of mouse (Ikedaetal.Proc.Natl.Acad.Sci.102 (32): 11331-11336,2005); Retinal progenitor cells (Klassenetal.Invest.Ophthal.Vis.Sci.45 (11): 4167-4175,2004) is generated by the retina of the 1st day mouse after being born; Bone marrow mescenchymal stem cell (Inoueetal.Exp.EyeRes.8 (2): 234-241,2007) in the RCS rat model of retinal degeneration; Retinal progenitor cells is produced by H1 human embryonic stem cell line, comprise ganglion cell, 0.01% sight sensor of expressing S-opsin or Visual purple of amacrine cell, wherein total cell, Beale's ganglion cells and horizontal cell (Lambaetal.Proc.Natl.Acad.Sci.10 (34): 12769-12774,2006); And by the multipotential stem cell (iPS) of human fibroblast's induction, thus produce retinal progenitor cells (Lambaetal.PLoSONE5 (1): e8763.doi:10.1371/journal.pone.0008763).These methods all can not produce the homogeneous population of sight sensor progenitor cell for transplanting or photoreceptor cell.These methods all can not produce the sight sensor progenitor cell of rod photoreceptor cell or cone cell function (such as can be perceiveed by the visual acuity of imparting improvement) or the homogeneous population of photoreceptor cell in display body.The supply of donor derived tissues (can isolate sight sensor and sight sensor progenitor cell by this tissue, such as corpse, fetal tissue and the animal lived) is restricted.Stem cell in vitro can Immortalization expanding, thus provides the potential source infinitely of non-donor derived cell for human treatment.Differentiation of stem cells becomes the homogeneous population of sight sensor progenitor cell or sight sensor can provide the non-donor derived cell of ample supply for the transplanting of retinal diseases and treatment.

Summary of the invention

In certain embodiments, the invention provides the substantially pure prepared product of sight sensor progenitor cell, it comprises: multiple sight sensor progenitor cell; And be applicable to the medium of the vigor keeping sight sensor progenitor cell.

In certain embodiments, the invention provides the prepared product of sight sensor progenitor cell, it comprises: the multiple cells containing at least 50% sight sensor progenitor cell; And be applicable to the medium of the vigor keeping sight sensor progenitor cell.

In certain embodiments, the invention provides the prepared product of sight sensor progenitor cell, it comprises: multiple sight sensor progenitor cell, it is not substantially containing multipotential stem cell, retinal ganglial cells, ripe sight sensor and/or amacrine cell, namely, comprise any one of these cells lower than 10%, even be more preferably lower than 5%, 2%, 1%, 0.1%, or even lower than 0.01% ocular multipotential stem cell, retinal ganglial cells, ripe sight sensor and/or amacrine cell; And be applicable to the medium of the vigor keeping sight sensor progenitor cell.

In certain embodiments, the invention provides the pharmaceutical preparations of the sight sensor progenitor cell being applicable to mammalian subject, it comprises: multiple sight sensor progenitor cell; And for keeping sight sensor progenitor cell transplantation to the medicine acceptable carrier of the vigor in mammalian subject.

In certain embodiments, the invention provides low temperature cellular preparations, it comprises: at least 10 ⁹individual sight sensor progenitor cell; And it is compatible with sight sensor progenitor cell and keep the cryopreservation system of this type of cell vigor after thawing.

In the preferred embodiment of above-mentioned prepared product, in described prepared product, the cell of at least 70% is PAX6+ and CHX10-in immunocytochemistry, and the mRNA transcript that (although alternatively) is detected as MASH1 by qPCR is positive; It is even furthermore preferable that in described prepared product, the cell of at least 80%, 90%, 95% or 98% is PAX6+ and CHX10-in immunocytochemistry, and the mRNA transcript that (although alternatively) is detected as MASH1 by qPCR is positive.

In certain embodiments, detected by qPCR, most sight sensor progenitor cell is positive for the mRNA transcript of Nr2e3, Tr β 2, ROR β and NRO.

In certain embodiments, as the immunoassay by secretory protein or by the detection of qPCR to mRNA transcript level, for retina neural progenitor cell, sight sensor progenitor cell expresses at least 2,3,4,5 or even more than 10 times be selected from one or more following protein: uPA, tenascin-C, CXCL16, CX3CL1 and chitinase 3 sample albumen 1.

In certain embodiments, sight sensor progenitor cell has replication, thus at least 10 are experienced in cell culture, 20,30,50 or the even population doublings of 100 times, and the 10th time, the 20th time, the 30th time, the 50th time or even the 100th multiplication time lower than 25% cell generation necrocytosis, aging or be divided into the cell that phenotype is non-sight sensor.

In certain embodiments, the Transferrins,iron complexes of sight sensor progenitor cell and/or Transferrins,iron complexes mRNA level in-site than glyceraldehyde 3 phosphate desaturase low at least 10,25,50 or even 75%.

In certain embodiments, sight sensor progenitor cell is identical in HLA genotype, and is preferably on genome identical.

In certain embodiments, the mean terminal Restriction Fragment Length (TRF) of sight sensor progenitor cell, it is longer than 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 10.5kb, 11kb, 11.5kb or even 12kb.

In certain embodiments, relative to the sight sensor that fetus is derivative, the content and/or the enzymic activity that participate in the protein of one or more processes following in sight sensor progenitor cell have statistically reduction significantly: the adjustment of (i) cell cycle and cell senescence; (ii) cellular energy and/or lipid metabolism; (iii) apoptosis.

In certain embodiments, the sight sensor derivative relative to fetus, relates to the content of cytoskeletal structure and the cytokinetic protein relevant with it and/or enzymic activity and has and statistically raise significantly in sight sensor progenitor cell.

In certain embodiments, sight sensor progenitor cell is applicable to give human patients.

In certain embodiments, sight sensor progenitor cell is applicable to the patient giving non-human animal doctor.

In the preferred embodiment of above-mentioned prepared product, sight sensor progenitor cell, derived from Mammals multipotential stem cell, particularly human pluripotent stem cell, is preferably selected from the multipotential stem cell of embryonic stem cell and induction.

In certain embodiments, sight sensor progenitor cell is originated by common multipotential stem cell to break up.

In certain embodiments, sight sensor progenitor cell maintains the plasticity-being divided into rod photoreceptor cell and cone cell.

In certain embodiments, sight sensor progenitor cell can transplanted in the subretinal space of ELOVL4-TG2 mouse, it will migrate to outer nuclear layer, and in ELOVL4-TG2 mouse, improve twilight vision and vision improvement ERG reaction.

In certain embodiments, sight sensor progenitor cell has phagocytic cell activity, such as, engulf sight sensor outer sections and/or the pHrodo of separation ^tMthe ability of RedE.coliBioParticles.

In certain embodiments, sight sensor progenitor cell secretes one or more neuroprotective factor.

In certain embodiments, be applicable to keep the medium of the vigor of sight sensor progenitor cell to be selected from developing medium, cryopreservation medium and be applicable to the biocompatibility injectable media of human patients injection.

In certain embodiments, sight sensor progenitor cell prepared product is without thermal source with without fungi.

The pharmaceutical preparations that another aspect provides the sight sensor being applicable to use in mammalian subject of this aspect, it comprises: the photosensory cell that multipotential stem cell is derivative, wherein be greater than 70%, 80%, 90%, the cell of 95% or even 98% in immunocytochemistry, be PAX6+, CHX10-and be Visual purple+and/or opsin+; And the medicine acceptable carrier for keeping photoreceptor cell to migrate to the vigor in mammalian subject.

This aspect another aspect provides pharmaceutical preparations, it comprises: retinal pigment epithelium, and sight sensor progenitor cell and/or photoreceptor cell; And the medicine acceptable carrier for keeping photoreceptor cell to migrate to the vigor in mammalian subject.The prepared product of cell (can mix with cell suspension, or the form of test kit, this test kit has the separate doses of co-administered cell) or the form of multi-layer cellular graft (being optionally arranged on biocompatible matrix or solid support) provide.When multi-layer cellular graft, RPE cell can provide with the form of the individual layer of individual layer, preferably polarization.

Another aspect of the present invention provides the disease and disorderly method that cause due to photoreceptor loss in treatment patient, and it comprises and gives this type of pharmaceutical preparations as herein described, the such as prepared product of sight sensor progenitor cell and/or photoreceptor cell.Described prepared product can be local injection, such as, be injected in the subretinal space of patient's eye, is injected in the vitreum of patient; Or system send or be passed to cell can in other body cavitys of sustainable existence.

The disease caused by the loss of sight sensor or disorder comprise macular degeneration (no matter the macular degeneration that such as age is relevant is early stage or late period) and retinitis pigmentosa.

In certain embodiments, the invention provides the method producing sight sensor progenitor cell, it comprises the following steps:

A ocular progenitor cell (be preferably the form of cell cluster, and preferably under the condition of low attachment or non-attachment) is cultivated by () in Neural Differentiation substratum is enough to make cell cluster form for some time of individual cells ball;

B () is under attachment condition, preferably in matrix (biological example stock support), cell ball is cultivated in Neural Differentiation substratum, until in culture most cell for being characterized by PAX6+, the retina neural progenitor cell of CHX10+ and SOX2-;

C () after this, between low attachment and the condition of non-attachment, one or many alternately changes culture condition, reach for some time that retina neural progenitor cell is enough to be formed single cell ball, then under attachment condition, cultivate the retina neural progenitor cell comprising cell ball, wherein continue alternately to change culture condition until most cell is sight sensor progenitor cell.

In preferred embodiments, such as in immunocytochemistry, ocular progenitor cell can be characterized by PAX6+, RX1+, OCT4-and NANOG-, and even more preferably can also be characterized by Six3+, Six6+, Lhx2+, Tbx3+, SOX2+ and nidogen+, as measured by the expression of markers characteristic in the cell that uses in immunostaining and/or flow cytometer or the other standards method of inspection.

In preferred embodiments, such as in immunocytochemistry, sight sensor progenitor cell is characterized by PAX6+ and CHX10-(such as can be measured by the expression of markers characteristic in the cell that uses in immunostaining and/or flow cytometer or the other standards method of inspection), and be more preferably also characterized by Mash1, Nr2e3, the mRNA transcript positive (as detected by qPCR) of Tr β 2, ROR β and NRO.

In preferred embodiments, sight sensor progenitor cell is characterized by and can be divided into photoreceptor cell when using retinoic acid therapy.

In preferred embodiments, sight sensor progenitor cell maintains the plasticity-being divided into rod photoreceptor cell and cone cell.

In preferred embodiments, when sight sensor progenitor cell is transplanted in the subretinal space of ELOVL4-TG2 mouse, it migrates to outside stratum nucleare, and in ELOVL4-TG2 mouse, for contrast (not injecting cell) ELOVL4-TG2 mouse, the reaction of twilight vision and vision improvement ERG can be improved.

In certain embodiments, attachment condition comprises the culture systems with surface, wherein cell can be attached on described surface, described system comprises attachment material, it for (only illustrating) or can comprise following one or more: polyester, polypropylene, polyalkylene, poly-fluorine vinylchlorid (polyfluorochloroethylene), polyvinyl chloride, polyfluoroethylene resin, polystyrene, polysulfones, urethane, polyethylene terephthalate, Mierocrystalline cellulose, glass fibre, ceramic particle, biomaterial scaffolds, poly (l-lactic acid), dextran, inert metal fiber, silica, mineral alkaline glass, borosilicate glass, chitosan or plant sponge (vegetablesponge).In some embodiments, attachment material is static electrification lotus.In certain embodiments, biomaterial scaffolds is extracellular matrix, such as collagen protein (such as IV type or type i collagen), the matrix that 804G is derivative, fibronectin, vitronectin, chondronectin, ln or Matrigel ^tM.In other embodiments, biomaterial is gelatin, alginate, PGA, fibrinogen or self-assembling peptides.

In certain embodiments, ocular progenitor cell and the retina neural progenitor cell thus obtained and sight sensor progenitor cell derived from multipotential stem cell, the multipotential stem cell of such as embryonic stem cell or induction.

In preferred embodiments, the sight sensor progenitor cell prepared product of gained, substantially not provide containing the form of multipotential stem cell, namely comprises lower than 10% multipotential stem cell, and be even more preferably lower than 5%, 2%, 1%, 0.1% or even lower than 0.01% multipotential stem cell.

In preferred embodiments, the sight sensor progenitor cell prepared product of gained is not substantially to provide containing the form of ocular progenitor cell and retina neural progenitor cell, namely comprise lower than any one of 10% these cells, even be more preferably lower than 5%, 2%, 1%, 0.1% or even lower than 0.01% ocular progenitor cell and retina neural progenitor cell.

In preferred embodiments, the cellular constituent of the sight sensor progenitor cell prepared product of gained relative to other types cell (namely, be not the cell of sight sensor progenitor cell) be at least 50% pure, and be preferably at least 75%, at least 85%, at least 95%, at least 99% or about 100% pure.

In certain embodiments, described method comprises the further step of cryopreservation sight sensor progenitor cell.Described cell is preferably freezing in cryopreservation medium, described cryopreservation medium with finally to thaw described frozen cell and washing described cell alternatively with after removing cryopreservation medium, described sight sensor keeps the cell viability (such as based on culture efficiency) of at least 25%, and be more preferably at least 50%, 60%, the cell viability of 70%, 80% or even at least 90% is compatible.

Multiple progenitor cell and photoreceptor cell can by cryopreservations.In some embodiments, sight sensor progenitor cell with spherical form by cryopreservation.

In certain embodiments, Neural Differentiation substratum (or being sometimes also called medium in this article) can comprise D-Glucose, penicillin, Streptomycin sulphate, GlutaMAX ^tM, N2 supplement, B27 supplement, MEM nonessential amino acid solution, and optionally comprise noggin.

Neural Differentiation substratum can comprise the reagent activating Notch approach, such as Notch part or antibody.

In certain embodiments, Neural Differentiation substratum can be substantially not containing the medium of serum, such as MEDII conditioned medium.In certain embodiments, Neural Differentiation substratum comprises DMEM/F12, FGF-2 and MEDII conditioned medium.In certain embodiments, Neural Differentiation substratum is the MEDII conditioned medium of about 10% to about 50%>.In certain embodiments, MEDII conditioned medium is HepG2 conditioned medium.MEDII substratum can comprise the extracellular matrix proteins of macromolecule.MEDII substratum can comprise the low molecular weight compositions containing proline(Pro).

In certain embodiments, Neural Differentiation substratum is comprise the cytodifferentiation environment substantially not containing serum lower than 5% serum.

In certain embodiments, Neural Differentiation substratum is not substantially containing LIF.

In addition, Neural Differentiation substratum can also comprise multiple supplement, such as B27 supplement (Invitrogen) and N2 supplement (also from Invitrogen).Except other constituents, B27 supplement also comprise SOD, catalase and other antioxidants (GSH), and the lipid acid of uniqueness, such as linolic acid, linolenic acid, Thioctic Acid.N2 supplement can by such as following mixture replacing: Transferrins,iron complexes (10g/L), Regular Insulin (500mg/L), progesterone (0.63mg/L), putrescine (1611mg/L) and selenite (0.52mg/L).

In above-mentioned and in some embodiment of embodiment, sight sensor progenitor cell is obtained by multipotential stem cell source differentiation, such as express OCT4, alkaline phosphatase, SOX2, SSEA-3, SSEA-4, the multipotential stem cell of TRA-1-60 and TRA-1-80 (such as but not limited to dry (ES) clone of embryo or dry (iPS) clone of inducing pluripotent), and be even more preferably and derive from common multipotential stem cell source.

In above-mentioned and in some embodiment of embodiment, sight sensor progenitor cell has and is greater than 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, the mean terminal Restriction Fragment Length (TRF) of 10.5kb, 11kb, 11.5kb or even 12kb.

In above-mentioned and in some embodiment of embodiment, prepared product is applicable to give human patients, and more preferably without thermal source and/or not containing inhuman animal product.

In above-mentioned and in some embodiment of embodiment, prepared product is applicable to give non-human animal doctor Mammals, such as but not limited to dog, cat or horse.

In an aspect, present disclose provides the method producing ocular progenitor cell, it comprises (a) and cultivate multipotential stem cell in retina inducing culture medium.Described multipotential stem cell can be the mankind.

Described retina inducing culture medium can comprise Regular Insulin.Described Regular Insulin can be the mankind.The concentration that described Regular Insulin can be about 5-50ug/ml human insulin or about 25ug/ml with concentration exists.Described retina inducing culture medium can comprise DMEM/F12, DMEM/ high glucose, or DMEM/knock-out.

Described retina inducing culture medium can comprise D-Glucose.Retina inducing culture medium can comprise about 450mg/mlD-glucose, or about 400 to about 500mg/mlD-glucose.

Retina inducing culture medium can comprise one or more microbiotic.Described microbiotic can comprise penicillin and/or Streptomycin sulphate, the concentration of penicillin is optionally about 0-100 unit/ml, the concentration of Streptomycin sulphate is optionally about 0-100 μ g/ml, and further, the concentration of penicillin is optionally about 100 units/ml, and the concentration of Streptomycin sulphate is optionally about 100 μ g/ml.

Retina inducing culture medium can comprise N2 supplement.Described N2 supplement can exist with the concentration of about 0.1 to 5% or about 1%.

Retina inducing culture medium can comprise B27 supplement.Described B27 supplement can exist with the concentration of about 0.05-2.0% or about 0.2%.

Retina inducing culture medium can comprise non-essential amino acid, MEM non-essential amino acid, glutamine or GlutaMAX ^tM.Described non-essential amino acid or MEM non-essential amino acid can exist with the concentration of about 0.1mM.

Retina inducing culture medium can comprise BMP signal transmission inhibitor.Described BMP signal transmission inhibitor can be selected from noggin (such as noggin polypeptide), Dorsomorphin, LDN-193189 or their arbitrary combination.

Retina inducing culture medium can comprise noggin, such as noggin polypeptide.Described noggin can exist with the concentration of about 5-100ng/ml, approximately 10-100ng/ml or about 50ng/ml.

In some embodiments, medium can comprise noggin, DKK1 and IGF-1.In some embodiments, medium can comprise 5ng/ml noggin, 5ng/mlDKK1 and 5ng/mlIGF-1.

Described multipotential stem cell can comprise Human ES cells, mankind iPS cell or mankind STAP cell.Described multipotential stem cell can without raise and/or without the condition of xenogenesis under cultivate, and/or optionally comprising Matrigel ^tMin the substrate of (Solubilised preparations obtained by Engelbreth-Holm-Swarm (EHS) mice sarcoma cell) and optionally cultivate on mTESR1 substratum, then cultivate in the described retina inducing culture medium comprising Regular Insulin.

The fresh retina inducing culture medium described in retina inducing culture Medium Replacement can be used every day.Described cultivation in step (a) can continue about 1-10 days, or about 2-7 days, or about 5-6 days.

Described method may further include (b) and cultivate described cell in Neural Differentiation substratum.Described Neural Differentiation substratum can comprise Neurobasal substratum.

Described Neural Differentiation substratum can comprise D-Glucose.Neural Differentiation substratum can comprise about 450mg/mlD-glucose, or about 400 to about 500mg/mlD-glucose.

Neural Differentiation substratum can comprise one or more microbiotic.Described microbiotic can comprise penicillin and/or Streptomycin sulphate, the concentration of penicillin is optionally about 0-100 unit/ml, the concentration of Streptomycin sulphate is optionally about 0-100 μ g/ml, and further, the concentration of penicillin is optionally about 100 units/ml, and the concentration of Streptomycin sulphate is optionally about 100 μ g/ml.

Neural Differentiation substratum can comprise N2 supplement.Described N2 supplement can exist with the concentration of about 0.1 to 5% or about 2%.

Neural Differentiation substratum can comprise B27 supplement.Described B27 supplement can with about 0.05-5.0%, and the concentration of about 0.05-2.0% or about 2% exists.

Neural Differentiation substratum can comprise non-essential amino acid, MEM non-essential amino acid, glutamine or GlutaMAX ^tM.Described non-essential amino acid or MEM non-essential amino acid can exist with the concentration of about 0.1mM.

Neural Differentiation developing medium can comprise BMP signal transmission inhibitor.Described BMP signal transmission inhibitor can be selected from: noggin (such as noggin polypeptide), Dorsomorphin, LDN-193189 and their arbitrary combination.

Neural Differentiation developing medium can comprise noggin, such as noggin polypeptide.Described noggin can exist with the concentration of about 10-100ng/ml or about 50ng/ml.

Described cell can be cultivated in described Neural Differentiation substratum about 10-60 days, approximately 15-35 days or about 24 days.

Described ocular progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.

Described ocular progenitor cell expresses mark PAX6 and/or RX1.Therefore, ocular progenitor cell can be PAX6 (+) and/or RX1 (+).Described ocular progenitor cell can be SIX3 (+), SIX6 (+), one or more in LHX2 (+), TBX3 (+) and/or nidogen (+).Described ocular progenitor cell can be one or more in SOX2 (+), OCT4 (-) and Nanog (-).Described ocular progenitor cell can be the mankind.

Described method may further include and makes described ocular ancestor cell differentiates become retina neural progenitor cell.

In one aspect of the method, present disclose provides the composition comprising ocular progenitor cell using method as herein described (such as method described in above-mentioned paragraph) to produce.In one aspect of the method, present disclose provides the composition comprising ocular progenitor cell, wherein said ocular progenitor cell is optionally the mankind's.

Described ocular progenitor cell can be the mankind.Described ocular progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.Described ocular progenitor cell expresses mark PAX6 and/or RX1.Therefore, ocular progenitor cell can be PAX6 (+) and/or RX1 (+).Described ocular progenitor cell can be SIX3 (+), SIX6 (+), one or more in LHX2 (+), TBX3 (+) and/or nidogen (+).Described ocular progenitor cell can be one or more in SOX2 (+), OCT4 (-) and Nanog (-).Described ocular progenitor cell can be cryopreservation.

In one aspect of the method, present disclose provides the method for the treatment of individuality in need, it comprises the composition (such as composition as herein described or the composition that uses method as herein described to produce) comprising ocular progenitor cell to described individuality.Described composition can be given in eyes, subretinal space or vein.Described individuality can have macular degeneration, comprise age relevant macular degeneration, and this type of macular degeneration can be early stage or late period.This type of individuality can have retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, and congenital retinal is malnutritive, Leber congenital amaurosis, retinal detachment, glaucoma or optic neuropathy.

In one aspect of the method, present disclose provides the method producing retina neural progenitor cell or sight sensor progenitor cell, it comprises (a) and cultivate ocular progenitor cell in Neural Differentiation substratum.Described Neural Differentiation substratum can comprise Neurobasal substratum.

Neural Differentiation developing medium does not optionally comprise the BMP signal transmission inhibitor that external source adds.Neural Differentiation substratum does not optionally comprise the noggin that external source adds, such as noggin polypeptide.

Step (a) can comprise: (i) cultivates ocular progenitor cell, until cell forms ball; And (ii) under attachment condition by described ball bed board.

Cell described in the flat board that step (i) can be included in low attachment is cultivated.Step (i) can be included in hanging drop cultivates described cell.Culturing cell can be divided into single cell suspension to be formed by machinery or enzyme process by the cultivation of step (i).Step (i) can continue 1-10,3-8 or about 5 days.

Step (ii) can comprise described ball is plated on Matrigel ^tMon.Step (ii) can comprise and being plated on ln or collagen protein by described ball.Step (ii) can continue until described culture converges.

Step (i) and (ii) can repeat in an alternating fashion.

Described cell can be cultivated in described Neural Differentiation substratum about 10-60 days, approximately 15-35 days or about 25 days.

Described retina neural progenitor cell can be obtained by described ocular ancestor cell differentiates, and can be present in described culture with the quantity increased.Described retina neural progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.

Described retina neural progenitor cell can express mark PAX6 and/or RX1.Therefore, neural progenitor cell can be PAX6 (+) and/or CHX10 (+).Described retina neural progenitor cell can be SOX2 (-).Described retina neural progenitor cell can be Tuj1 (+) or Tuj1 (-).

Described cell can be cultivated in described Neural Differentiation substratum about 10-330 days, approximately 15-300 days, approximately 10-100 days, approximately 15-100 days or about 100 days.

Described sight sensor progenitor cell is obtained by described retina neural ancestor cell differentiates, and can be present in described culture with the quantity increased.Described sight sensor progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.

Described sight sensor progenitor cell can be PAX6 (+) and/or CHX10 (-).Described sight sensor progenitor cell can express mark Nr2e3, Tr β 2, Mash1, one or more in ROR β and/or NRO, and therefore can be Nr2e3 (+) and/or Tr β 2 (+) and/or Mash1 (+) and/or ROR β (+) and/or NRO (+).

Described cell can be cultivated at least about 130 days in described Neural Differentiation substratum, at least about 160 days, at least about 190 days or longer, sight sensor progenitor cell described thus shows the ability being divided into cone cell of reduction or does not have this ability, and keeps the ability forming rod photoreceptor cell.

Described method may further include and makes described sight sensor ancestor cell differentiates become sight sensor.

Described ocular progenitor cell can be obtained by pluripotent stem cell differentiation, such as ES cell, iPS cell or STAP cell, and this multipotential stem cell (such as ES cell, iPS cell or STAP cell) can be optionally the mankind's.

In one aspect of the method, described retina neural progenitor cell can be the mankind's.

In one aspect of the method, present disclose provides the composition comprising retina neural progenitor cell produced according to any method as herein described, such as described in the preceding paragraph method.In one aspect of the method, present disclose provides the composition comprising retina neural progenitor cell, it is optionally the mankind's.

Described retina neural progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.

Described retina neural progenitor cell can express PAX6 and/or CHX10 mark, and therefore can be PAX6 (+) and/or CHX10 (+).Described retina neural progenitor cell can be SOX2 (-).Described retina neural progenitor cell can be Tuj1 (+) or Tuj1 (-).

Described retina neural progenitor cell can cryopreservation.

In one aspect of the method, present disclose provides the method for individuality for the treatment of and having this to need, it comprises the composition (such as composition as herein described or the composition that uses method as herein described to produce) comprising retina neural progenitor cell to described individuality.Described composition can be given in eyes, subretinal space or vein.Described sight sensor progenitor cell can be the mankind.This type of individuality can have macular degeneration, comprise age relevant macular degeneration, and this type of macular degeneration can be early stage or late period.This type of individuality can have retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, and congenital retinal is malnutritive, Leber congenital amaurosis, retinal detachment, glaucoma or optic neuropathy.

In one aspect of the method, present disclose provides the composition comprising sight sensor progenitor cell produced according to method as herein described, such as described in the preceding paragraph method.In one aspect of the method, present disclose provides the composition comprising sight sensor progenitor cell, described cell is optionally the mankind's.

Described sight sensor progenitor cell can comprise at least 50%, at least 75%, at least 85%, at least 95% of cell in described culture, and at least 99% or about 100%.

Described sight sensor progenitor cell can be PAX6 (+) and/or CHX10 (-).Described sight sensor progenitor cell can express Nr2e3, Tr β 2, Mash1, one or more in ROR β and/or NRO mark, and therefore can be Nr2e3 (+) and/or Tr β 2 (+) and/or Mash1 (+) and/or ROR β (+) and/or NRO (+).

Described sight sensor progenitor cell can be cryopreservation.

In one aspect of the method, present disclose provides the method for individuality for the treatment of and having this to need, it comprises the composition (such as composition as herein described (such as in above-mentioned paragraph) or use the composition of the method generation such as described in aforementioned paragraphs described herein) comprising sight sensor progenitor cell to described individuality.Described composition can be given in eyes, subretinal space or vein.This type of individuality can have macular degeneration, comprise age relevant macular degeneration, and this type of macular degeneration can be early stage or late period.This type of individuality can have retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, and congenital retinal is malnutritive, Leber congenital amaurosis, retina shedding, glaucoma or optic neuropathy.

In one aspect of the method, present disclose provides the method producing photoreceptor cell, it comprises (a) and cultivate sight sensor progenitor cell in photoreceptor differentiation substratum.Described photoreceptor differentiation substratum can comprise Neurobasal substratum.

Described photoreceptor differentiation substratum can comprise D-Glucose.Photoreceptor differentiation substratum can comprise about 450mg/mlD-glucose, or about 400 to about 500mg/mlD-glucose.

Susceptor division culture medium can comprise one or more microbiotic.Described microbiotic can comprise penicillin and/or Streptomycin sulphate, the concentration of penicillin is optionally about 0-100 unit/ml, the concentration of Streptomycin sulphate is optionally about 0-100 μ g/ml, and further, the concentration of penicillin be optionally about 100 units/ml and the concentration of Streptomycin sulphate optionally for about 100 μ g/ml.

Photoreceptor differentiation substratum can comprise N2 supplement.Described N2 supplement can exist with the concentration of about 0.1 to 5% or about 2%.

Photoreceptor differentiation substratum can comprise B27 supplement (such as formula number 080085-SA).Described B27 supplement can with about 0.05-5.0%, and the concentration of about 0.05-2.0% or about 2% exists.

Photoreceptor differentiation substratum can comprise non-essential amino acid, MEM non-essential amino acid, glutamine or GlutaMAX ^tM.GlutaMAX ^tMfor Ala-Gln, it is the stabilized form of L-glutaminate.Described non-essential amino acid or MEM non-essential amino acid can exist with the concentration of about 0.1mM.

Described photoreceptor differentiation substratum can comprise forskolin.Described forskolin can exist with the concentration of about 1-100 μM or about 5 μMs in photoreceptor differentiation substratum.

Described photoreceptor differentiation substratum can comprise BDNF.Described BDNF can exist with the concentration of about 1-100ng/ml or about 10ng/ml in photoreceptor differentiation substratum.

Described photoreceptor differentiation substratum can comprise CNTF.Described CNTF can exist with the concentration of about 1-100ng/ml or about 10ng/ml in photoreceptor differentiation substratum.

Described photoreceptor differentiation substratum can comprise LIF.Described LIF can exist with the concentration of about 5-50ng/ml or about 10ng/ml in photoreceptor differentiation substratum.

Described photoreceptor differentiation substratum can comprise DATP.Described DATP can exist with the concentration of about 1-100 μM or about 10 μMs in photoreceptor differentiation substratum.

Described sight sensor progenitor cell can be obtained by retina neural ancestor cell differentiates, and described retina neural progenitor cell is optionally the mankind.Described photoreceptor cell can be the mankind.

In some embodiments, use vitamin A acid and taurine to carry out pre-treatment to sight sensor progenitor cell in ND substratum, then cultivate in photoreceptor differentiation substratum.Vitamin A acid can use with the concentration of about 100-1000ng/ml, and taurine can use with the concentration of about 20-500 μM.In some embodiments, this culturing step can carry out about 1-2 week.In some cases, within every 2 days, a medium (such as changing half) can be changed.Then, Medium Replacement is become to lack the ND substratum of vitamin A acid and taurine, and described cell can cultivate in addition approximately 1-2 week, or until they converge.

In one aspect of the method, present disclose provides the composition comprising the sight sensor produced according to the method for (such as described in the preceding paragraph) described herein, described cell is optionally the mankind's.

Described photoreceptor cell can be PAX6 (-).Described photoreceptor cell can comprise at least 50%, at least 75% of cell in described culture, and at least 85%, at least 95%, at least 99% or about 100%.Described photoreceptor cell can be cryopreservation.

In one aspect of the method, present disclose provides the method for the treatment of the individuality having this to need, it comprises the composition (composition such as herein described in (such as in above-mentioned paragraph) or the composition using the method herein described in (such as in above-mentioned paragraph) to produce) comprising photoreceptor cell to described individuality.Described composition can be given in eyes, subretinal space or vein.This type of individuality can have macular degeneration, comprise age relevant macular degeneration, and this type of macular degeneration can be early stage or late period.This type of individuality can have retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, and congenital retinal is malnutritive, Leber congenital amaurosis, retina shedding, glaucoma or optic neuropathy.

In another embodiment, the present invention relates to the sight sensor progenitor cell (PRPC) in people source or the substantially pure prepared product of photoreceptor cell (PR), be preferably the non-sight sensor progenitor cell for syntaxy or photoreceptor cell, it originates from raises at l cell the cell that platform does not grow.Such as prepared product can be that 85%-95% is pure.In one embodiment, the present invention relates to the method for the substantially pure prepared product of preparation people source PRPC or PR, which omits the needs of being raised platform derived cell by l cell.Use method of the present invention to substitute feeding system and can produce the higher photoreceptor cell of homogeneity, such as 75%-100% or 85%-95%.In addition, can also carry out the differentiation of the stem cell without feeder layer when not introducing exogenous inducing factors, this is from being better than prior art in fact.But, add the differentiation that noggin can accelerate stem cell alternatively, even if need not differentiation phase be being carried out.The sight sensor progenitor cell of gained can be characterized by the PAX6 positive (PAX6 (+)) and CHX10 feminine gender (CHX10 (-)) uniquely in immunocytochemistry.

Accompanying drawing is sketched

When requiring and pay required expense, the copy of the open and color drawings (scheming) of this patent or patent application will be provided by government department more.

Fig. 1: in the cell broken up under different conditions, the real-time PCR analysis of the transcript of ocular transcription factor.

Fig. 2: the morphology of noble cells.(A) the 1st day after cytodifferentiation, the cell of margin of colonies was cylindricality (arrow).(B) after differentiation the 10th day, border cell becomes large and flat (arrow), centrocyte little and fine and close (arrow).(C) Rose spline structure was formed at the 21st day.

Fig. 3: after differentiation starts, 21 days time, cultured cells expresses ocular transcription factor.(A) coexpression of PAX6 (green) and RX1 (redness), on the accompanying drawing of colored version clearly.(B) cell coexpression PAX6 and RX1 of 93%, as shown in Two-color flow cytometry analysis.(C) nidogen (redness) of cell expressing.(D) SOX2 (redness) of cell expressing.In (C) and (D), DAPI (blueness) labeled cell core.(E) RT-PCR of the transcript of ocular transcription factor analyzes: RX1, PAX6, LHX2, SIX3, SIX6, TBX3 and SOX2.

Fig. 4: after differentiation starts, 30 days time, cultured cells expresses retina neural progenitor cell marker thing.(A) morphology of cell.Be plated on Matrigel ^tMafter upper, neurone by cell aggregation to external migration (arrow).Less epithelioid cell's (arrow) is observed around cell aggregation.(B) the little figure in top is the neuronic phase difference image of migration; The little figure in below is the Tuj1 (redness) of migration neuron expression.(C) PAX6 (redness) of cell coexpression and CHX10 (green), is obvious by the accompanying drawing of colored version.

Fig. 5: after differentiation starts, cultured cells 3 months time.(A) morphology of cell.(B) cell expressing PAX6, but do not express CHX10, and be obvious by the accompanying drawing of colored version, it shows some cells and dyes redness, but does not dye green.(C) expression of recoverin (Recoverin) is limited in the tenuigenin of cell paste, and the accompanying drawing of colored version is obvious.(D) in retina neural progenitor cell (RNP) and sight sensor progenitor cell (being expressed as PhRP), the Real time RT-PCR analysis of the transcript of Visual purple, opsin and recoverin.

Fig. 6: the cell expressing photoreceptor cell mark of differentiation.(A) Visual purple (redness) of cell expressing, (B) Visual purple (redness) and recoverin (green), (C) opsin (green), and (D) phosphodiesterase 6A α subunit (PDE6a) (redness).DAPI (blueness) labeled cell core.The expression of these marks is obvious by the accompanying drawing of colored version.

Fig. 7: in ELOVL4 transgenic mice, the schematic diagram of zooscopy.

Fig. 8: after subretinal injection cell, recorded twilight vision ERG intensity-reaction functions 1 month time.The twilight vision a ripple (upper little figure) obtained by the ELOVL4-TG2 mouse giving PBS (black line) or sight sensor progenitor cell (being expressed as PhRP, grey lines) and the stimulus intensity curve of b ripple (lower little figure).*, p<0.001 (compared with PBS).

Fig. 9: after systemic injection cell, recorded twilight vision ERG intensity-reaction functions 1 month time.The twilight vision a ripple (upper little figure) obtained by the ELOVL4-TG2 mouse of the sight sensor progenitor cell (PhRP-RA) giving PBS, sight sensor progenitor cell (being expressed as PhRP) or retinoic acid treatments and the stimulus intensity curve of b ripple (lower little figure).The untreated mouse of blank expression.#, p<0.001 (compared with PBS).

Figure 10 A-B: between 1 month to 2 months after Transplanted cells, sight sensor progenitor cell systemic injection has recovered the function of rod photoreceptor cell.Obtained by the ELOVL4-TG2 mouse of the sight sensor progenitor cell (PhRP-RA) giving PBS (PBS) or retinoic acid treatments, when injecting after cell 1 month and 2 months the twilight vision ERG amplitude of a ripple (A) and b ripple (B).

Figure 10 C: after systemic injection cell, the twilight vision ERG intensity-reaction functions of record 2 months time.The twilight vision a ripple (the little figure in top) obtained by the ELOVL4-TG2 mouse of the sight sensor progenitor cell (PhRP-RA) giving PBS or retinoic acid treatments and the stimulus intensity curve of b ripple (the little figure in below).The untreated mouse of blank expression.Baseline is the level of record 4 weeks time.*, p<0.001 (compared with PBS).

Figure 11: obtained by the ELOVL4-TG2 mouse of the sight sensor progenitor cell (PhRP-RA) giving PBS or retinoic acid treatments, when injecting after cell 1 month and 2 months the vision improvement ERG amplitude of a ripple (the little figure in top) and b ripple (the little figure in below).*, p<0.001 (compared with when PBS2 month).

Figure 12: by untreated ELOVL4-TG2 mouse (blank) and give that the mouse of sight sensor progenitor cell (PhRP-RA) of PBS, sight sensor progenitor cell (PhRP) or retinoic acid treatments obtains, after injecting cell 1 month and 2 months time the whole foveal region of retina thickness measured by OCT.

Figure 13: (A) obtained by the ELOVL4-TG2 mouse of the susceptor progenitor cell (the little figure in right side) giving PBS (the little figure in left side) and retinoic acid treatments, the presentation graphics of the retina HE dyeing when injecting after cell 2 months.ONL is outer nuclear layer.INL inner nuclear layer.(B) by untreated ELOVL4-TG2 mouse (blank) and give that the mouse of sight sensor progenitor cell (PhRP-RA) of PBS or retinoic acid treatments obtains, after injecting cell retina ONL thickness quantitative when 2 months.

Figure 14: the schematic diagram of zooscopy in RCS rat.

Figure 15: after the sight sensor progenitor cell that Transplanted Human DX-like centers is derivative, the preservation of host's photoreceptor cell.Use that DAPI dyes, under P90 retinal slice: (A), in control rats, outer nuclear layer (ONL) is reduced to 0-1 layer.(B) after intravenous injection cell, save ONL cell in RCS rat, its degree of depth is 2-4 cell.(C) in the RCS rat accepting intravitreal injection cell, save ONL cell, its degree of depth is 3-5 cell.INL is inner nuclear layer; GL is ganglion-cell layer.

Figure 16: after the sight sensor progenitor cell that Transplanted Human DX-like centers is derivative, the preservation of host's rod photoreceptor cell photoreceptor cell outer sections (OS).That dye for Visual purple (green), under P90 retinal slice.(A) in control rats, rod photoreceptor cell OS (arrow) is lost completely.(B) after intravenous injection sight sensor progenitor cell, the expression (arrow) of Visual purple in the OS of host's rod photoreceptor cell photoreceptor cell in RCS rat retina.(C) after transplanting sight sensor progenitor cell in vitreum, the expression (arrow) of Visual purple in the OS of host's rod photoreceptor cell photoreceptor cell in RCS rat retina.The expression of Visual purple is obvious in the accompanying drawing of colored version.

Figure 17: after the sight sensor progenitor cell that Transplanted Human DX-like centers is derivative, the preservation of host cone cell photoreceptor cell outer sections (OS).That dye for opsin (green), under P90 retinal slice.(A) in control rats, cone cell OS (arrow) is lost completely.(B) after intravenous injection sight sensor progenitor cell, the expression (arrow) of opsin in the OS of host cone cell photoreceptor cell in RCS rat retina.(C) after transplanting sight sensor progenitor cell in vitreum, the expression (arrow) of opsin in the OS of host cone cell photoreceptor cell in RCS rat retina.The expression of opsin is obvious in the accompanying drawing of colored version.

Figure 18: the rod photoreceptor cell photoreceptor cell migrating to the sight sensor ancestor cell differentiates one-tenth maturation that the Human ES cells in the vitreum of RCS rat derives.For Visual purple (being green in A), that recoverin (for green in B) dyes, under P90 retinal slice.Use anti-HuNU antibody labeling human cell (redness).DAPI marks all core.Expression and the use DAPI dyeing of Visual purple and recoverin are obvious in the accompanying drawing of colored version.

Figure 19 illustrates the group method for sight sensor research and development in embodiment 1-2, and the medium used in each step of this process is shown further.

Figure 20 illustrates the time course of photoreceptor cell and the research and development of sight sensor progenitor cell in embodiment 1-2.

Figure 21 illustrates the composition of developing medium and the medium supplement used in an embodiment.

Figure 22 illustrates the gene expression pattern of ESC, ocular progenitor cell, retina neural progenitor cell, sight sensor progenitor cell and photoreceptor cell in the vitro differentiation process of human pluripotent stem cell.

Figure 23 provides flow cytometric histograms, it illustrates at 37 DEG C and 4 DEG C, hES-RPE and hES-sight sensor progenitor cell with contrast (lifeless matter particle) and compare pHrodo ^tMthe phagocytotic relative extent of RedE.coliBioParticles (Invitrogen) fluorescence biological particle.The histogram display of sight sensor progenitor cell, be similar to RPE cell, when the physiology associated temperature of described sight sensor progenitor cell by the relative non-permitted temperature change to 37 DEG C of 4 DEG C, the intensity of fluorescent signal increases, and shows that sight sensor progenitor cell can phagotroph particle.Biological particles is the surrogate of outer sections and glassy membrane wart of coming off in eyes.

Detailed Description Of The Invention

The invention provides the method for generating photoreceptor cell (PRC) and sight sensor progenitor cell (PRPC).These methods relate to the vitro differentiation of early progenitor cell, and wherein said progenitor cell comprises multipotential stem cell, ocular (EF) progenitor cell and retina neural progenitor cell.Method provided herein can use above-mentioned progenitor cell (comprising stem cell) colony any one as parent material.

Present invention further contemplates and generate photoreceptor cell (PRC) and sight sensor progenitor cell (PRPC) outward (namely by primary ocular (EF) progenitor cell and retina neural progenitor somatic, primary cell refers to the cell obtained by study subject, instead of the cell obtained by the vitro differentiation of more jejune progenitor cell).

The research and development of sight sensor are undertaken by a large amount of development, and each stage can be defined by phenotype (such as by the expression map of mark) and/or function.These research and development schematically show in fig. 22.Multipotential stem cell vitro differentiation becomes EF progenitor cell, and it is divided into retina neural progenitor cell then, itself so that be divided into photosensory cell progenitor cell, it is divided into photoreceptor cell again.

As used herein, progenitor cell refers to such cell: it keeps mitotic division, and can produce more progenitor cells of identical or more limited differentiation capability, or can be divided into last destiny clone eventually.Term progenitor cell and precursor can exchange use.The cell in each stage will discussed in more detail in these stages herein.

Sight sensor progenitor cell (photoreceptorprogenitorcell, photoreceptorprogenitor) provided herein and photoreceptor cell may be used in multiple body and in vitro method.Such as sight sensor progenitor cell may be used for treating amphiblestroid situation in vivo, includes but not limited to macular degeneration and retinitis pigmentosa.Sight sensor progenitor cell and photoreceptor cell may be used in screening assay in vitro, thus the therapeutic of qualification presumption or prophylactic treatment material standed for.

Invention further provides the sight sensor progenitor cell and photoreceptor cell that are obtained by method as herein described.Obtain sight sensor progenitor cell by vitro differentiation multipotential stem cell or their differentiation offspring (such as ocular progenitor cell) and photoreceptor cell ocular progenitor cell itself can be obtained by the vitro differentiation of multipotential stem cell, or they can for deriving from the primary ocular progenitor cell of study subject.

The invention provides and not yet to be obtained by primary source or not obtainable sight sensor progenitor cell populations and photoreceptor cell colony.These colonies can be homogeneities or close to homogeneity in their entocyte.Such as, in this types of populations, the cell of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or about 100% can be sight sensor progenitor cell.As another example, in this types of populations, the cell of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or about 100% can be photoreceptor cell.In these colonies, these cells can be single haplotypes.Such as they can be HLA couplings.These cells in these colonies can be that heredity is upper consistent.

The disclosure based on the direct differentiated progenitor cells of disclosed method (such as but not limited to multipotential stem cell) ability and the prepared product of substantially pure (or homogeneity) of multiple cell colony is provided.As used herein, directed differentiation refers to that part makes this type of progenitor cell populations be divided into required clone owing to being supplied to the factor of progenitor cell or other stimulator or to its differentiation, thus avoids being divided into other unwanted cells systems and the clone of contaminative potentially thus.Method provided herein drives such as pluripotent stem cell differentiation to become ocular progenitor cell, and does not produce embryoid body (EB).As mentioned below, EB is three-dimensional cell bunch, it can be formed in the atomization of multipotential stem cell (including but not limited to embryonic stem cell (ES)), and usually comprise mesoderm, ectoderm and endoderm cell system cell, comprise progenitor cell.The three-dimensional person's character of EB can create the environment being different from and being formed in non-EB based method as herein described, comprises different cell-ECM and interacts and different cell-ECM signal transmission.In addition, the reagent that the external source that the cell in EB can not all accept comparable amount adds, the differentiation factor such as existed in medium around, and this can cause differentiation events different in the growth course of EB and decision.

On the contrary, the present invention's cultural method of cultivating progenitor cell without the need to and preferably avoid the formation of EB.And these methods can culturing cell under the following conditions: for cell provides the contact equal with surrounding medium, comprise the factor in this type of medium.Such as cell with the individual layer be attached on culture surface or can grow close to the form of individual layer.

The ability that all or most progenitor cell contacts with roughly equal degree and its surrounding medium and the factor correspondingly in this type of medium makes these progenitor cells be divided into similar degree in the similar time.This similar divergaence time table for progenitor cell populations shows that this type of cell is synchronized.In addition, in some cases, cell can be cell cycle synchronization.This homochronousness obtains such cell colony, and their cellularity is homogeneity or close to homogeneity.Such as method as herein described can produce cell colony, and wherein the cell of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or about 100% is paid close attention to specific cells.The cell paid close attention to can such as be defined in phenotype by the expression of in cell or extracellular mark.The cell paid close attention to can be ocular progenitor cell, neural retina progenitor cell, sight sensor progenitor cell or photoreceptor cell.

As used herein, most cell refers at least 50%, and according to embodiment, can comprise at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or the cell of about 100%.

At this type of cell colony by when being used for the treatment of in vivo or preventing object, the purity that method of the present invention can be used to obtain is particularly important.The ability obtaining the colony of high cell purity avoids implements another kind of operation, and such as enrichment or selection step, these steps can cause unnecessary loss cell.When cell colony is less or cell quantity is limited, the colony obtaining high cell purity is particularly important.

Definition: as defined herein, provide singulative to be for illustrative purposes, odd number can also be used for the plural form of described phrase.Be intended to the traditional definition of complementary terms to give a definition, they can understand by those of ordinary skill.

" the substantially pure prepared product of sight sensor progenitor cell (PRPC) ".As used herein, this phrase is the prepared product (such as wrapping celliferous composition) of phalangeal cell, and wherein said cell is at least 75% pure, or is preferably at least 85% pure, and at least 95% is pure, or about 85% to 95% is pure.Such as can pass through to measure in prepared product and express the level of one or more marks (those marks of such as PRPC comprise those marks or other marks known in the art of identifying in this application) relative to the next quantitative purity of ratio of the sum (such as by detecting the cell of expressing or not expressing one or more described marks) of cell in prepared product.In addition, optionally can also detect the expression of the mark indicating non-PRPC cell, promote the detection and/or quantitatively of described cell thus.Operable illustrative methods includes but not limited to fluorescence activated cell sorting (FACS), immunohistochemical method, hybridization in situ and other suitable methods known in the art.Optionally, the mensuration of purity can be carried out, and the unvital cell existed in prepared product need not be taken into account.

" the substantially pure prepared product of the photoreceptor cell (PR) in people source ".As used herein, this phrase refers to such cellular preparations (such as wrapping celliferous composition), and wherein said cell is at least 75% pure, or is preferably at least 85% pure, and at least 95% is pure, or about 85% to 95% is pure.Such as can pass through to measure in prepared product and express the level of one or more marks (those marks of such as PR comprise those marks or other marks known in the art of identifying in this application) relative to the next quantitative purity of ratio of the sum (such as by detecting the cell of expressing or not expressing one or more described marks) of cell in prepared product.Optionally can also detect the expression of the mark indicating non-PR cell, promote the detection and/or quantitatively of described cell thus.Operable illustrative methods includes but not limited to fluorescence activated cell sorting (FACS), immunohistochemical method, hybridization in situ and other suitable methods known in the art.Optionally, the mensuration of purity can be carried out, and the unvital cell existed in prepared product need not be taken into account.

" embryoid body " refer to multipotential cell (such as iPSC or ESC) agglomerate or bunch, it can such as, by cultivating multipotential cell and formed, in the substrate of low attachment or in " hanging drop " under non-attachment condition.In these cultures, multipotential cell can be formed the cell of called after embryoid body agglomerate or bunch.See Itskovitz-Eldoretal., MolMed.2000Feb; 6 (2): 88-95, it is incorporated to herein by reference of text.Usually, embryoid body formed at first the solid of multipotential cell agglomerate or bunch, and through after a period of time, some embryoid bodies start to comprise the chamber that fluid is full of, and the former is called " simply " EB in the literature, and the latter is called " cyst " embryoid body.

Term " embryonic stem cell " (ES cell or ESC) uses in this article as in this area.This term comprises by the derivative cell of human blastocyst or morular inner cell mass, comprises as this clone through those of serial passage.ES cell can derivatively by the fertilization of ovum and sperm obtain, and use DNA, consideration convey moves, monogenesis or derivatively to obtain by being created on HLA region and having homozygotic ES cell.In addition, ES cell is derived from zygote, blastomere or blastocyst stage mammiferous embryonic derived cell, wherein said zygote, blastomere or blastocyst stage mammiferous embryo be merged by sperm and ovum, consideration convey moves, monogenesis, androgenesis, or by chromatinic restructuring and subsequently the chromatin of restructuring is introduced in the cytoplasmic membrane for generation of cell produce.Embryonic stem cell (no matter they source or for generation of their ad hoc approach) can identify based on following aspect: (i) is divided into the ability of the cell of all 3 germinal layers; (ii) expression of at least OCT4 and alkaline phosphatase; And (iii) produces teratomatous ability when migrating in immunodeficiency type animal.The embryonic stem cell that can use in embodiments of the invention includes but not limited to Human ES cells's (" ESC " or " hES cell "), such as MA01, MA09, ACT-4, No.3, H1, H7, H9, H14 and ACT30 embryonic stem cell.Other exemplary cells system comprises NED1, NED2, NED3, NED4, NED5 and NED7.In addition, see NIH hESC registry form.Operable exemplary human embryonic stem cell line is MA09 cell.Described by having in Klimanskaya, etal. (2006) " HumanEmbryonicStemCelllinesDerivedfromSingleBlastomeres. " Nature444:481-485 before the separation of MA09 cell and preparation.The Human ES cells that exemplary according to the present invention uses can derive according to GMP standard and keep.

Term " ES cell " can not be inferred and should be inferred as this cell by destroying embryo and generating.On the contrary, multiple method is available and may be used for generating ES cell, and does not destroy embryo, such as human embryos.Such as ES cell can be generated by the single blastomere derived from embryo, and its mode is similar to the extraction of the blastomere for implanting front gene diagnosis (PGD).The example of this type of clone comprises NED1, NED2, NED3, NED4, NED5 and NED7.Operable exemplary human embryonic stem cell line is MA09 cell.Described by having in Klimanskaya, etal. (2006) " HumanEmbryonicStemCelllinesDerivedfromSingleBlastomeres. " Nature444:481-485 before the separation of MA09 cell and preparation.In addition, see Chungetal.2008, CellStemCell, 2:113.All these clones are all generate when not destroying embryo.

As used herein, term " multipotential stem cell " includes but not limited to that the stem cell of tissue derived, embryonic stem cell, embryonic derived stem cell, induced multi-potent stem cells and stimulation trigger versatility (STAP) cell obtained, and no matter the method for derivative multipotential stem cell is how.In addition, described term also comprises and has the function of cell mentioned above and the multipotential stem cell of phenotypic characteristic, and no matter for how generating the method for this type of cell.Multipotential stem cell is functionally defined as following stem cell, and it is: (a) can induce teratoma when migrating in immunodeficiency type (SCID) mouse; B () can be divided into the cell type (such as can be divided into ectoderm, mesoderm and endoblastic cell type) of all 3 germinal layers; And (c) expresses one or more marks (such as expressing OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1 etc.) of embryonic stem cell.In certain embodiments, multipotential stem cell is expressed and is selected from one or more following marks: OCT4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.Example method as known in the art can generate exemplary multipotential stem cell.Exemplary multipotential stem cell comprises the embryonic stem cell of the ICM derived from blastocyst stage embryo; And the embryonic stem cell (optionally not destroying the residuum of embryo) of one or more blastomeres derived from division stage or morula stage embryo.This embryonic stem cell-like can be generated by fetal material, and described fetal material is by fertilization or produced by asexual means (comprising SCNT (SCNT), monogenesis and androgenesis).Other exemplary multipotential stem cells comprise the induced multi-potent stem cells (iPSC) generated by recombinant cell (by expressing the combination of the factor, being referred to herein as the reprogrammed factor).Can use fetus, birth after, newborn infant, teenager or be grown up somatocyte to generate iPSC.

In certain embodiments, may be used for being become by reprogramming of somatic cells the factor of multipotential stem cell to comprise such as OCT4 (being commonly referred to OCT3/4), the combination of SOX2, c-Myc and KLF4.In other embodiments, may be used for being become by reprogramming of somatic cells the factor of multipotential stem cell to comprise the combination of such as OCT4, SOX2, Nanog and Lin28.In certain embodiments, in somatocyte, express at least 2 kinds of reprogrammed factors, thus successfully reprogrammed somatocyte.In other embodiments, in somatocyte, express at least 3 kinds of reprogrammed factors, thus successfully reprogrammed somatocyte.In other embodiments, in somatocyte, express at least 4 kinds of reprogrammed factors, thus successfully reprogrammed somatocyte.In other embodiments, identify other the reprogrammed factor, and be used alone or use with one or more known reprogrammed combinations of factors, thus reprogramming of somatic cells is become multipotential stem cell.Induced multi-potent stem cells functionally defines, and comprises the cell (integrative vector, non-integrated vector, chemical means etc.) using any one of multiple method to carry out reprogrammed.Multipotential stem cell can be genetic modification or adopt other means to modify, thus increase life-span, effect, go back to the nest, prevent or reduce homoimmune reaction, or the cell broken up by this class multipotential cell (such as sight sensor, sight sensor progenitor cell, rod photoreceptor cell, cone cell etc., and the cell of other types as herein described, such as, in embodiment) in send the required factor.

" multipotential stem cell of induction " (iPS cell or iPSC) can be produced by the protein transduction of the reprogrammed factor in somatocyte.In certain embodiments, at least 2 kinds of reprogrammed factors are transduceed in somatocyte, thus successfully reprogrammed somatocyte.In other embodiments, at least 3 kinds of reprogrammed factors are transduceed in somatocyte, thus successfully reprogrammed somatocyte.In other embodiments, at least 4 kinds of reprogrammed factors are transduceed in somatocyte, thus successfully reprogrammed somatocyte.

Multipotential stem cell can derive from any species.Embryonic stem cell is successfully derivative in multiple species and the mankind of such as mouse, non-human primate, and embryonic stem cell-like cell produces from other species multiple.Therefore, those skilled in the art can generate embryonic stem cell and embryonic derived stem cell by any species, includes but not limited to the mankind, non-human primate, rodent (mouse, rat), ungulates (ox, sheep etc.), dog (family dog and wild dog), cat (domestic cat and wildcat, such as lion, tiger and African cheetah), rabbit, hamster, gerbil jird, squirrel, cavy, goat, elephant, panda (comprising giant panda), pig, racoon, horse, zebra, marine mammal (dolphin, whale etc.) etc.In certain embodiments, described species are species in imminent danger.In certain embodiments, described species are current extinct specie.

Similarly, iPS cell can derive from any species.These iPS cells have used mouse and human cell successfully to generate.In addition, use embryo, fetus, newborn infant and adult group to knit and successfully generate iPS cell.Therefore, people can use the donorcells deriving from any species easily to generate iPS cell.Therefore, people can generate iPS by any species, include but not limited to the mankind, non-human primate, rodent (mouse, rat), ungulates (ox, sheep etc.), dog (family dog and wild dog), cat (domestic cat and wildcat, such as lion, tiger and African cheetah), rabbit, hamster, goat, elephant, panda (comprising giant panda), pig, racoon, horse, zebra, marine mammal (dolphin, whale etc.) etc.In certain embodiments, described species are species in imminent danger.In certain embodiments, described species are current extinct specie.

Any budding almost any somatocyte can be used as starting point to generate the multipotential stem cell of induction.Such as cell can derive from embryo, fetus, newborn infant, teenager or adult donor.Operable exemplary somatocyte comprises inoblast, the skin flbroblast such as obtained by skin samples or examination of living tissue sample, the synovial cell obtained by synovial tissue, foreskin cells, cheek cell or lung fibroblast.Although the easy utilization that skin and cheek provide suitable cell and the source easily obtained, can use almost any cell.In certain embodiments, somatocyte is not inoblast.

Can by expressing in somatocyte or inducing the expression of one or more reprogrammed factors to produce the multipotential stem cell of induction.Somatocyte can be inoblast, such as skin flbroblast, synovioblast or lung fibroblast, or non-fibroblastic somatocyte.Can by making at least 1,2,3,4,5 kinds of reprogrammed factor expressions (such as by viral transduction, integration or non-integrated vector etc.) and/or contact with these factors (such as use protein transduction domains, electroporation, microinjection, cationic amphiphilic molecule, and double-layer of lipoid merges, washing composition penetrates) carry out reprogrammed somatocyte.The reprogrammed factor can be selected from OCT3/4, SOX2, NANOG, LIN28, C-MYC and KLF4.The expression of inducing the reprogrammed factor can be contacted with at least one reagent of reprogrammed factor expression (such as little organic molecule reagent) can be induced by making somatocyte.

Other exemplary multipotential stem cell comprises by the induced multi-potent stem cells of expressing or the combinational expression of inducible factor (" the reprogrammed factor ") generates reprogramming of somatic cells.IPS cell can be obtained by cell bank.In the production of noble cells, preparation iPS cell can be initial step.In order to generate PHRPS or the photoreceptor cell of Organization Matching, the material of the donor deriving from particular patient or coupling can be used specifically to generate iPS cell.IPSC can produce by substantially not having in intended recipient immunogenic cell, such as, produce by autogenous cell or by the cell with intended recipient histocompatibility.

In addition, combined method can also be used to come reprogramming of somatic cells, wherein express the expression (such as using little organic molecule) of the reprogrammed factor (such as using virus vector, plasmid etc.) and the induction reprogrammed factor in the described method.Such as, virus vector (such as retroviral vector or lentiviral vectors) can be used in somatocyte, to express the reprogrammed factor by infecting.In addition, can use nonconformable carrier (such as plasmid episomal) in somatocyte, express the reprogrammed factor.For example, see Yuetal., Science.2009May8; 324 (5928): 797-801, the document is incorporated to herein in full with way of reference.When the nonconformable carrier of use expresses reprogrammed because of the period of the day from 11 p.m. to 1 a.m, carrier can be used to carry out transfection, conversion or electroporation and the factor described in cells to somatocyte.Such as in mouse cell, using integrated virus vector to express 4 kinds of factors (OCT3/4, SOX2, C-MYC and KLF4) is enough to reprogrammed somatocyte.In human cell, using integrated virus vector to express 4 kinds of factors (OCT3/4, SOX2, NANOG and LIN28) is enough to reprogrammed somatocyte.

Once the reprogrammed factor is at cells, just this cell can be cultivated.Through the regular hour, the cell with ES feature appears in culture dish.Can cell be selected, and based on the morphology of such as ES or carry out succeeding transfer culture based on the expression of selectable or detectable mark.Described cell can be cultivated, thus produce the cell culture (these cells are the iPS cell of presumption) being similar to ES cell.

In order to prove the versatility of iPS cell, in the test of one or more versatility, check described cell.Described cell is checked in expression such as ES cell sign thing; When by Transplanted cells to SCID mouse time, for generation teratomatous ability evaluate cell; The ability producing the cell type of all 3 germinal layers for differentiation evaluates cell.Once obtain versatility iPSC, just may be used for producing cell type disclosed herein, such as sight sensor progenitor cell, photoreceptor cell, rod photoreceptor cell, cone cell etc., and other cell type as herein described, such as, described in embodiment.

Stimulate the multipotential stem cell of versatility (STAP) cell for producing by using semilethal stimulation (being such as exposed to low pH) to make reprogramming of somatic cells triggering and obtain.Reprogrammed need not move in somatocyte or to somatocyte and carries out genetic manipulation by consideration convey.Can with reference to Obokataetal., Nature, 505:676-680,2014.

" stem cell " is used in reference in this article and can breeds and/or be divided into mature cell and be the pluripotent cell in people source alternatively.

" adult stem cell " refers to the pluripotent cell by the separate tissue of adult, and can comprise bone marrow stem cell, cord blood stem cell and fat stem cell, and is people source.

" retina " refers to the neurocyte of eyes, and it is divided into 3 stratum nucleares, is made up of sight sensor, horizontal cell, Beale's ganglion cells, amacrine cell, Muller cell and ganglion cell.

" precursor cell " refers to the cell that can be divided into last destiny clone eventually.In embodiments of the invention, " ocular progenitor cell " is obtained by embryonic stem cell or induced multi-potent stem cells differentiation, and it expresses mark PAX6 and RX1.In embodiments of the invention, " retina neural progenitor cell " refers to and breaks up by the embryonic stem cell of express cell mark PAX6 and CHX10 or induced multi-potent stem cells the cell obtained.In embodiments of the invention, " sight sensor progenitor cell " refer to broken up by embryonic stem cell or induced multi-potent stem cells and express mark PAX6 and do not express the cell of mark CHX10 (that is, CHX10 (-)).These cells are at retina neural progenitor cell phase transient expression CHX10, but when cytodifferentiation becomes the sight sensor progenitor cell phase, the expression of CHX10 is closed.In addition, " sight sensor " can refer to be broken up by embryonic stem cell or induced multi-potent stem cells and in express cell mark Visual purple or 3 kinds of cone cell opsins any one and optionally express the postmitotic cells of rod photoreceptor cell or cone cell cGMP phosphodiesterase.In addition, sight sensor can also express mark recoverin, and it finds in sight sensor.Sight sensor can be rod photoreceptor cell and/or cone cell sight sensor.

" sign " of disease is as used herein, and broad sense refers to any exception that can find in the inspection of patient, that show disease; Contrary with symptom (it is that the subjectivity of disease indicates), it is the objective instruction of disease.

" symptom " of disease is as used herein, and broad sense refers to that patient experiences and shows any ill phenomenon of disease or deviate from normal in structure, function or sensation.

" therapy ", " treatment ", " therapeutic ", " treatment " or " therapeutics " are as used herein, and broad sense refers to disease therapy, stop or the development of slow down disease or its clinical symptom, and/or palliate a disease, disease or its clinical symptom are reduced.Therapy covers prevention, stops, treats, cures, remedies, slows down, slows down and/or palliate a disease, the sign of disease and/or symptom.Therapy covers the sign and/or symptom that slow down occurent disease indication and/or symptom in patient.In addition, therapy also covers " prevention " and " prevention ".Prevention comprises the disease preventing from occurring after disease treatment in patient, or reduces incidence or the seriousness of disease in patient.When being used for the treatment of, term " minimizing " broad sense refers to the minimizing of the clinical meaning of sign and/or symptom.Therapy comprises treatment and sends out or the sign that recurs and/or symptom again.Therapy contains but is not limited to always get rid of the appearance of sign and/or symptom, and reduces the existing sign of existing sign and/or symptom and elimination and/or symptom.Therapy comprises treatment of chronic diseases (" maintenance ") and acute illness.Such as treatment comprises treatment or prevents sending out or recurring of sign and/or symptom.

According to the present invention and the situation that uses one or more prepared products provided in this article treat thus includes but not limited to macular degeneration, comprise the macular degeneration that the age is correlated with, and this type of macular degeneration can be early stage or late period.Other situations medicable include but not limited to retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal is malnutritive, Leber congenital amaurosis, retina shedding, glaucoma, optic neuropathy and affect the wound of eyes.

Cell sign thing: may be used for express assessment exemplary cells mark comprise following these: PAX6, RX1, SIX3, SIX6, LHX2, TBX3, SOX2, CHX10, nidogen, TRbeta2, NR2E3, NRL, MASH1, RORbeta, recoverin, opsin, Visual purple, rod photoreceptor cell and cone cell cGMP phosphodiesterase (it can assess on protein and/or mRNA level in-site) are (see FischerAJ, RehTA, DevNeurosci.2001; 23 (4-5): 268-76; Baumeretal., Development.2003Jul; 130 (13): 2903-15, Swaroopetal., NatRevNeurosci.2010Aug; 11 (8): 563-76, AgathocleousandHarris, Annu.Rev.CellDev.Biol.2009.25:45-69, every a document is all incorporated to herein in full with way of reference).Mark identifier as described in routine use the same in document with this area, in particularly relevant to this content (wherein these genetic identifier are as described herein) technical field, described document can comprise and can comprise and sight sensor, rod photoreceptor cell, cone cell, photoreceptor differentiation, sight sensor progenitor cell, Neural Differentiation, neural stem cell, multipotential stem cell and the relevant document of other field as herein described.In addition, mark is generally the mankind, unless made contrary explanation in such as context.Traditional immunocytochemistry or traditional PCR method can be used to identify cell sign thing, and the technology of these methods is that those of ordinary skill in the art are known.

Cell culture medium: in embodiments of the invention, cell stores in various kinds of cell developing medium, propagation or differentiation.Retina induction medium is used for making stem cell form ocular progenitor cell.Retina induction medium can comprise D-Glucose, penicillin, Streptomycin sulphate, N2 supplement (such as 0.1-5%), B27 supplement (such as 0.005 to 0.2%), MEM nonessential amino acid solution (and optionally comprising Regular Insulin and/or noggin) may reside in DMEM/F12 (Invitrogen) or similar media base.Such as retina induction medium at least can comprise Regular Insulin.In addition, the concentration of Regular Insulin can change or increase, and this can promote the survival of cell and/or the generation of noble cells.The concentration of such as Regular Insulin can change in certain scope, and monitoring survival and/or differentiation, thus qualification can improve one or both insulin concentration of these characteristics.It is believed that and add noggin not necessarily, but observe the expression that can increase ocular transcription factor.

DMEM/F12 is provided, Neurobasal substratum, the composition of N2 serum supplement and B27 serum supplement in Figure 21.It should be understood that the application of these specific substratum and supplement is contained in the present invention, or comprise, substantially by or become the substratum that is grouped into or supplement by these.

Method as herein described can use human Factor, such as human tau albumen, human insulin etc.

Noggin is the BMP inhibitor of secretion, it is reported, this inhibitor with high-affinity in conjunction with BMP2, BMP4 and BMP7, thus can block the activity of TGF 'beta ' family.SB431542 is small molecules, it is reported that it passes through to block the phosphorylation of ACTRIB, TGF β R1 and ACTRIC acceptor and suppresses TGF β/Activin/Nodal.SB431542 is considered to the versatility unstable networks that Activin-and Nanog-can be made to mediate, and the trophoderm, mesoderm and the endoderm cell's destiny that suppress BMP to induce by the signal blocking endogenous Activin and BMP.Estimate that the reagent with one or more activity above-mentioned can substitute or strengthen the function of noggin and/or SB431542, such as, when during they are for the content of the disclosure method.Such as the applicant estimates that protein tau albumen and/or small molecules SB4312542 can be substituted by one or more inhibitor (the following 3 kinds of target zones of impact any or all of) or strengthen: the combination 1) stoping part and acceptor; 2) activation (such as Dorsomorphin) of acceptor is blocked; And 3) suppress SMAD intracellular protein/transcription factor.The exemplary potential suitable factor comprises natural secretor type BMP inhibitor Chordin (it blocks BMP4) and Follistatin (it blocks Activin) and their analogue or stand-in.Other exemplary factors of effect of dummy head albumen can comprise and use dominant negative receptor or closed BMP2, BMP4, and/or the blocking antibody of BMP7.In addition, for the phosphorylation blocking acceptor, reported that Dorsomorphin (or Compound C) has similar effect to stem cell.In addition, can also use soluble inhibitors (such as SIS3, it is 6; 7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo-[2,3-b] pyridin-3-yl-propyl-2-enoyl-))-1,2; 3,4-tetrahydroisoquinoline, is smad 3specific inhibitor and SIS3), the process LAN of one or more inhibitor SMAD (such as SMAD6, SMAD7, SMAD10) or for acceptor SMAD (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8/9) one of RNAi realize the suppression of SMAD protein.Estimate that the another kind of combinations of factors being applicable to generate neural progenitor cell comprises leukaemia inhibitory factor (LIF), GSK3 inhibitor (CHIR99021), compd E (gamma-secretase inhibitors XXI) and TGF beta inhibitor SB431542 (have shown it effectively for generating stem cell of neural crest (Lietal., ProcNatlAcadSciUSA.2011May17 before; 108 (20): 8299-304) mixture).The exemplary factor in addition can comprise the derivative of SB431542, such as, comprise one or more that add or different substituting group, similar functional group etc. and have similar inhibiting molecule to one or more SMAD protein.Such as can by pluripotent cell be contacted with the described factor, and (such as characteristic gene is expressed to monitor the ocular progenitor cell phenotype adopted, comprise the expression of mark as herein described, expression etc. with the reporter gene of ocular progenitor cell promotor coupling) or form cell type disclosed herein (such as retina neural progenitor cell, sight sensor progenitor cell, rod photoreceptor cell progenitor cell, cone cell and/or rod photoreceptor cell) ability, identify the suitable factor or the combination of the factor.

Preferably, use the cell described in the process of retina induction medium or cultivate described cell in retina induction medium, then using Neural Differentiation culture medium culturing.Neural Differentiation substratum is used for making ocular progenitor cell produce retina neural progenitor cell.Neural Differentiation substratum can comprise D-Glucose, penicillin, Streptomycin sulphate, GlutaMAX ^tM, N2 supplement, B27 supplement, MEM nonessential amino acid solution, and optionally comprise noggin.In addition, Neural Differentiation substratum can also be used for making retina neural progenitor cell produce sight sensor progenitor cell, but does not comprise noggin.Use Neural Differentiation substratum (being optionally supplemented with vitamin A acid and taurine), (it optionally can comprise D-Glucose, penicillin, Streptomycin sulphate, GlutaMAX then to use photoreceptor differentiation substratum (Invitrogen) ^tM, N2 supplement, B27 supplement (such as formula number 080085-SA), and add forskolin, BDNF, CNTF, LIF and DATP) for making sight sensor progenitor cell produce photoreceptor cell.Such as photoreceptor differentiation substratum can comprise Triiodothyronine, such as, with the amount existed in medium before or different or higher amount.Such as described medium can comprise the Triiodothyronine that external source adds.In an exemplary embodiment, photoreceptor differentiation substratum can comprise BDNF, the one in CNTF and DATP, 2 kinds or all 3 kinds, such as BDNF, CNTF, DATP, BDNF and CNTF, CNTF and DATP, BDNF and DATP, or all 3 kinds of BDNF, CNTF and DATP, this medium optionally can comprise Neurobasal substratum and/or optionally can comprise Triiodothyronine.

The constituent of Neural Differentiation substratum is as follows: N2:1% (the every 100ml of 1mlN2), B27:2% (the every 100ml of 2mlB27), and noggin: 50ng/ml.

After cell all becomes ocular progenitor cell, just no longer need noggin.

Embryonic stem cell (ESC), adult stem or induced multi-potent stem cells (iPS): ESC used herein, adult stem or iPS cell can bred without on feeding system, such as, at Matrigel ^tMin (Solubilised preparations obtained by Engelbreth-Holm-Swarm (EHS) mice sarcoma cell) or another kind of matrix.In addition or alternatively, described pluripotent cell can be cultivated being selected from following matrix: ln, fibronectin, vitronectin, proteoglycan, nidogen, collagen protein, collagen protein I, collagen protein IV, collagen protein VIII, Suleparoid, Matrigel ^tM(Solubilised preparations obtained by Engelbreth-Holm-Swarm (EHS) mice sarcoma cell), CellStart, mankind's basement membrane extract and their arbitrary combination.Described matrix can comprise Matrigel ^tM(Solubilised preparations obtained by Engelbreth-Holm-Swarm (EHS) mice sarcoma cell), consisting of or substantially consisting of.Stem cell does not form embryoid body in culture, and this is better than prior art.Described cell is divided into ocular progenitor cell under the condition lacking exogenous factor.In one embodiment, ESC is divided into ocular progenitor cell under noggin exists.

Ocular progenitor cell (EFPC): EFPC is divided into the cell of PAX6 (+) and RX1 (+) by ESC, adult stem or induced multi-potent cell (iPC).In addition, EFPC can also be SIX3 (+), SIX6 (+), LHX2 (+), TBX3 (+) nidogen (+) and/or SOX2 (+), OCT4 (-) and NANOG (-).In retina inducer substance, be divided into EFPC, wherein said matrix can comprise DMEM/F12, D-Glucose, penicillin, Streptomycin sulphate, N2 supplement, B27 supplement, MEM non-essential amino acid and Regular Insulin.At the 5th day, when the cells reached confluency, cell is changed in Neural Differentiation substratum.Preferably, implement the step producing EFPC, then in Neural Differentiation substratum hereinafter described, cultivate pluripotent cell, because observed the vigor that above-mentioned culture condition adversely can affect pluripotent cell.

Retina neural progenitor cell (RNPC): under the condition that exogenous factor lacks, RNPC is obtained by EFPC differentiation.RNPC is PAX6 (+) and CHX10 (+).Cell in this state can be Tuj1+ or Tuj1-.Optionally, described method can comprise Tuj1+ or the Tuj1-cell in enrichment or purifying this period, and/or purifying or removing Tuj1+ cell consumingly, and/or purifying or removing Tuj1-cell (such as even lacking the cell that low-level can detect expression) consumingly, and carry out follow-up, method steps by one or the other in these colonies.In one embodiment, add noggin, to promote that EFPC is divided into RNPC.In Neural Differentiation substratum, be divided into RNPC, wherein said Neural Differentiation substratum can comprise Neurobasal substratum (Invitrogen), D-Glucose, penicillin, Streptomycin sulphate, GlutaMAX ^tM, N2 supplement, B27 supplement and MEM nonessential amino acid solution.Can add noggin, its final concentration is 5-100 μ g/ml.

Sight sensor progenitor cell (PhRPC): under noggin lacks and in Neural Differentiation substratum, PhRPC can be obtained by RNPC differentiation.PRPC is PAX6 (+) and CHX10 (-).In one embodiment, the PRPC of 60%, 70%, 80%, 85%, 90% or 95% is PAX6 (+) and CHX10 (-).In addition, PRPC can also be Nr2e3 (+), Tr β 2 (+), Mash1 (+), ROR β (+) and/or NRO (+).The existence of CHX10 shows that Beale's ganglion cells is, but in the method for the invention, PRPC has been divided into photoreceptor cell system, and therefore it does not have CHX10 in this period.Described cell can grow into spheroid or neural ball (such as on low attached flat board, or optionally on Hanging drop culture thing, under low-gravity environment or under other suitable culture condition).

Sight sensor (PR): in the process of two steps differentiation, PR can be broken up by PhRPC and obtain: 1) add the Neural Differentiation substratum 2 weeks with vitamin A acid and taurine; And 2) add photoreceptor differentiation substratum.See embodiment 2.

PR can be Visual purple (+), recoverin (+), PE6a (+) or opsin (+).Described opsin can be any one of cone cell opsin.For cone cell or rod photoreceptor cell, PR can be bipotential.The exemplary light susceptor produced by method of the present invention can be PAX6-, and this may be contrary with the photoreceptor cell claimed described before some.As hereinafter as described in exemplary, there is the differentiation method of 2 steps: 1) add ND substratum, vitamin A acid and taurine 2 weeks; And 2) using photoreceptor differentiation substratum, the method further illustrates in following working examples.

In an exemplary embodiment, described method is by every 1x10 ⁶individual initial pluripotent cell can produce 40-60 1,000,000 EFPC, 60-90x10 ⁶individual RNPC or 0.5-1x10 ⁹individual PhRPC.

In an exemplary embodiment, can by Transplanted cells in the rat having this to need, other animal models (such as yctalopia or colour blindness) of such as RCS rat or disease, and test by optomotor response inspection, ERG, luminance threshold record and/or Optic center blood flow detection the impact that sight function is produced.

Application and purposes

Selective mechanisms

The invention provides the method for screening plurality of reagents, wherein said reagent can regulate and control the differentiation of retinal progenitor cells.In addition, it can also be used for the treatment reagent finding to support and/or save ripe sight sensor, and wherein said sight sensor is generated by retinal progenitor cells in culture.For object of the present invention, " reagent " intention includes but not limited to biological or chemical compound, such as simple or complicated organic or inorganic molecule, peptide, protein (such as antibody), polynucleotide (such as antisense) or ribozyme.Can synthesize the compound of enormous amount, such as polymkeric substance (such as polypeptide and polynucleotide) and the anthropogenics based on multiple core texture, and these materials are also contained in term " reagent ".In addition, multiple natural origin can be provided for the compound screened, such as plant or animal extracts etc.Although it should be understood that and always do not clearly state, described reagent can be used alone, or combinationally uses from another kind of reagent (having identical or different biological activity with the reagent that screening of the present invention is identified).

In order to implement screening method in vitro, can the cell colony that separation be obtained as described herein.When described reagent is composition (small molecules such as mentioned above) except DNA or RNA, described reagent can directly add in cell or join in the developing medium for adding.As apparent to those skilled in the art, " effectively " amount must be added, this amount can be measured by rule of thumb.When described reagent is polynucleotide, can directly add this reagent by use particle gun or electroporation.Alternatively, gene delivery vehicle or additive method mentioned above can be used to be inserted in cell by this reagent.Positive and negative control can be tested to prove the activity of claiming of medicine or other reagent.

Neural sensation retinal structure

Sight sensor progenitor cell, and optionally be may be used for generating neural sensation retinal structure by the photoreceptor cell of its differentiation.Such as contemplated by the invention the generation of multi-layer cellular structure, wherein said multi-layer cellular structure is made up of retinal pigment epithelium (RPE) cell and photoreceptor cell (or sight sensor progenitor cell).These structures may be used for medicine screening, as the model of disease, or as pharmaceutical preparations or in pharmaceutical preparations.In the latter case, pharmaceutical preparations can be RPE-sight sensor graft, it can be arranged on biocompatibility solid support or matrix (being preferably matrix or the upholder of biological absorbable), and this upholder or matrix can be implanted as " patch ".

In order to further illustrate, the biocompatibility upholder for cell can be the biodegradable polyester film upholder for retinal progenitor cells.Biodegradable polyester can for being suitable for use as any biodegradable polyester of substrate or support, and wherein said substrate or support are for supporting propagation and the differentiation of retinal progenitor cells.Polyester should be able to form film, is preferably the film of microtexture, and if for tissue or Transplanted cells, should be biodegradable.Poly(lactic acid) (PLA) is comprised for suitable biodegradable polyester of the present invention, polylactide, the homopolymer of polyhydroxyalkanoatefrom and multipolymer (such as poly butyric ester (PHB), hydroxybutyric acid and hydroxyl pentanoate copolymer (PHBV), poly butyric ester is hydroxycaproic ester (PHBHx) altogether, poly butyric ester is Hydroxyoctanoic acid ester (PHBO) and poly butyric ester hydroxy stearic acid ester (PHBOd) altogether altogether), polycaprolactone (PCL), polyesteramide (PEA), aliphatic copolyester (such as poly butylene succinate (PBS) and poly-succinic/tetramethylene adipate (PBSA)), aromatic copolyester.High and low-molecular-weight polyester can be used, replacement with unsubstituted polyester (block, branching or random), and the mixture of polyester and blend.Preferably, biodegradable polyester is polycaprolactone (PCL).

In certain embodiments, biocompatibility upholder is poly-(sub-p-Xylol) polymkeric substance, such as polyphenylene ethyl N, polyphenylene ethyl D, polyphenylene ethyl-C, polyphenylene ethyl AF-4, polyphenylene ethyl SF, polyphenylene ethyl HT, polyphenylene ethyl VT-4 and polyphenylene ethyl CF, and most preferably polyphenylene ethyl-C.

Usually, can use known technology that polymer supports is formed film.The thickness benefits ground of film is about 1 micron to about 50 microns, and thickness is preferably about 5 microns.The surface of film can be smooth, or the surface of film can be partly or entirely have microtexture.Suitable surface tissue comprises such as small groove or small post (micro-post).Described film can cut or shaping, thus forms the suitable shape for implanting.

Can by RPE and/or photoreceptor cell or sight sensor progenitor cell together or successively (such as after formation RPE layer, bed board photoreceptor cell or sight sensor progenitor cell) be directly plated on film, thus form biocompatible scaffold.Alternatively, suitable coating material coat polymers film can be used, such as poly-D-Lys, poly-L-Lysine, fibronectin, ln, collagen protein I, collagen protein IV, vitronectin and Matrigel ^tM.Can by described plating cells to any required density, but the RPE cell of individual layer (RPE individual layer) is preferred.

Therepic use

In addition, the invention provides in the method needing to substitute or repair in the patient for the treatment of photoreceptor cell, it comprises and gives pharmaceutical preparations to patient, and this pharmaceutical preparations comprises sight sensor progenitor cell of the present invention, by its derivative sight sensor or their combination.As described herein, pharmaceutical preparations can be the cell suspension or the cell that form portable tissue in vitro.In many cases, cell can be given ill or retrograde amphiblestroid subretinal space.But; because sight sensor progenitor cell of the present invention also has neuroprotective; so described cell can be used partly, but outside retina (such as in vitreum), or by reservoir or systems communicate to other parts of body.

In the various diseases that pharmaceutical preparations of the present invention may be used for causing vision system to worsen and disorder, comprise the disease that retinal degeneration is relevant.Agingly can cause this type of disease and disorder, so wherein show and lack the damage or disease that identifiable design is the basic source worsened.Technician can understand for diagnosing this type of morbid state and/or checking the existing method of this type of known sign damaged.In addition, in the vision system of animal, document provides the information of sufficient decline of being correlated with about the age or deterioration.Term " retinal degeneration relevant disease " refers to by born or acquired retinal degenerative or the abnormal any disease caused.The example of the disease that retinal degeneration is correlated with comprises retinal dysplasia, retinal degeneration, aging type macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal is malnutritive, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy and wound.

In addition or alternatively, the deterioration of vision system integral part (such as neural sensation retina) can be caused by damage, such as to the wound of vision system (such as eyes) itself, the damage of enemy or brain, or more usually to the wound of body.Some this type of damage known is the damage of being correlated with at the age, and namely along with the age, the possibility of damage or frequency increase.The example of this type of damage comprises tears retinal, macular hole, preretinal membrane and retinal detachment, each all may occur in the animal at any age, but the frequency that more may occur or occur in aging animal (comprising geriatric animals healthy in other respects) is higher.

In addition, the deterioration of vision system or its integral part can also be caused by disease.The disease etc. that the multiple age that these diseases comprise affects vision system is correlated with.This type of disease in geriatric animals than the possibility occurred in young animals and/or frequency higher.Vision system (comprising such as sensory nerve layer of retina) may be affected and the example of the disease causing it to worsen is following various ways: the retinitis, optic neuritis, macular degeneration, proliferative or non-proliferative diabetic retinopathy, diabetic macular edema, Progressive symmetric erythrokeratodermia neurodeatrophia, Progressive symmetric erythrokeratodermia retinal degeneration, acute acquired retinal degeneration, immune-mediated retinopathy, retinal dysplasia, choroidoretinitis, retinal ischemia, retinal hemorrhage is (before retina, in retina and/or under retina), hypertensive retinopathy, retinal inflammation, retinal edema, cancer eye or retinitis pigmentosa.

Some diseases mentioned above some animal specificity often, such as companion animals, such as dog and/or cat.Some of them disease is listed general, and namely can have and be permitted the eurypalynous retinitis or retinal hemorrhage, therefore, some diseases is not caused by the specific virulence factor of one, and describe type or the result of disease more.Can cause the one or more integral parts of vision system that decline or the numerous disease that worsens occur and can have former and secondary or farther effect to the vision system of animal.

Advantageously, pharmaceutical preparations of the present invention may be used for shortage or the reduction of compensating light receptor cell function.The example of the retinal dysfunction of retinal stem cells colony of the present invention and method treatment can be used to include but not limited to: sight sensor is degenerated (such as in retinitis pigmentosa, Progressive cone dystrophy, cone cell-rod photoreceptor cell and/or rod photoreceptor cell-Progressive cone dystrophy, and occur in macular degeneration); Retinal detachment and retinal injury; The light injury caused by laser or daylight; Macular hole; Macular edema; Yctalopia and colour blindness; The ischemic retinopathy caused by diabetes or angiemphraxis; Due to the retinopathy that precocity/premature labor causes; Infection state, such as the CMV retinitis and toxoplasmosis; Inflammatory condition, such as uveitidies; Tumour, such as cancer eye and eye malignant melanoma; And for replacing inner retina neurone, it is affected in optic neuropathy (comprising glaucoma, Traumatic optic neuropathy change, radiativity optic neuropathy and retinopathy).

In an aspect, in a patient in need for the treatment of, the symptom of retinitis pigmentosa can be treated or alleviate to described cell.In yet another aspect, in the patient needing this treatment, the symptom of macular degeneration can be treated or alleviate to described cell, macular degeneration (moist or dryness), Stargardt disease, degeneratio,maculae myopia etc. that such as the age is relevant.For all these treatments, described cell can be autologous or allos for patient.In one aspect of the method, cell of the present invention can combine with other treatment and give.

Retinitis pigmentosa (RP) refers to that one group of various heredity eye is disorderly, it is characterized by the Progressive symmetric erythrokeratodermia vision loss caused due to degenerating gradually of sight sensor.In the U.S., estimate at 100000 people and suffer from RP.Under a gauge, the classification of this group disorder is based on the Clinical symptoms the most often observed in these patients.The mark of RP is that yctalopia and indirect vision decline, and retinal vessel narrows, and pigment migrates in retina by the retinal pigment epithelium destroyed, and forms the agglomerate usually closing on the various size of retinal vessel.

Usually, first patient notices due to the loss of rod photoreceptor cell sight sensor and causes being difficult to see at night; Then, remaining cone cell sight sensor becomes the main dependence of sight function.But through in a few years or decades, cone cell also degenerates, and causes the Progressive symmetric erythrokeratodermia of vision to lose.In most RP patient, ocular defect starts from midperiphery, between 30 ° to 50 ° of distance fixation.Defect area expands gradually, forms vision island around with in constrained central field of vision (so-called tubular visual field).When the visual field be confined to 20 ° or lower and/or central vision is 20/200 or lower time, patient becomes jural blind person.

Hereditary pattern shows that RP can chain with X (XLRP), autosomal dominant (ADRP) or recessive (ARRP) pattern transmission.In these three kinds of hereditary forms of RP, ADRP is the slightest.These patients are until 60 years old and greater age still maintain good central vision usually.On the contrary, the patient suffering from XLRP form disease has been just blind legally at 30 to 40 years old usually.But, in the patient of RP suffering from homologous genes type, the seriousness of paresthesia epilepsy and older amplitude variation.Even when inferring that all affected members have identical transgenation, in identical family, this change is also obvious.At present, the sudden change of many induction RP has been described.In current identified gene, many genes encoding sight sensor specific proteins, some of them protein conducts relevant with the light in rod photoreceptor cell, the such as subunit of Visual purple, cGMP phosphodiesterase and the Ca of cGMP-gate ²⁺passage.In each clone gene, have been found that various mutations.Such as when rhodopsin gene, in ADRP patient, 90 kinds of different sudden changes are identified.

Regardless of concrete sudden change, be the degeneration gradually due to cone cell for the most important vision loss of RP patient.In many cases, the protein causing the sudden change of RP to affect even is not expressed in cone cell; Best example is Visual purple-rod photoreceptor cell specific visual pigment.Therefore, cone cell loss may be the indirect consequence of rod photoreceptor cell specific mutant.The ability substituting impaired sight sensor provides method for treating this disease.

Age relevant macular degeneration (AMD) causes the Progressive symmetric erythrokeratodermia of central vision to lose, and the prevailing reason of vision loss occurs its people being above 55 years old.Potential pathology are that sight sensor is degenerated.Multinomial research has disclosed the risk that gene, cardiovascular disorder, environmental factor (being such as exposed to cigarette and light) and nutrition reason contribute to developing into AMD.The different loss that RPE degeneration is poured into upper strata sight sensor and lower floor's choroid.When RPE fails and the upper strata photoreceptor cell secondary causing it to supply is lost, visual acuity loss or visual field losses.The ability substituting RPE and photoreceptor cell provides a kind of means for treating established AMD.

Macular degeneration is broadly divided into 2 types.In exudative-neovascular form or in " moist " AMD (they occupy 10% of all situations), issue the misgrowth of angiogenic at macula lutea.Which has been formed the retina lower network of choroid cardiovascularization, this is usually relevant with inter-retinal hemorrhage, subretinal fluid, retinal pigment epithelium detachment and hyperpigmentation.Finally, this mixture shrinks and leaves obviously outstanding scar at rear pole place.These vascular leakage fluids and blood, in retina, cause the damage to sight sensor thus.Moist AMD often rapid progress, and serious damage can be caused; Just can the rapid loss of generative center vision through only some months.

The AMD case of residue 90% is atrophic macular degeneration (dry form), wherein at macular area, pigment disorder can occur, but the macula lutea scar do not given prominence at macular area and do not have hemorrhage or seepage.In these patients, retinal pigment epithelium (RPE) fades away, thus causes the ischemic area of limited range.Due to RPE disappear after photoreceptor loss, affected retinal area has little or does not have sight function.The vision loss caused by dryness AMD occurs more gradually in experience process for many years.These patients keep some central visions usually, but this loss can seriously must be enough to damage the mission performance needing to watch details.

When suitable age and clinical discovery lose with visual acuity, the visual field or other sight functions, described situation is categorized as AMD usually.Usually, if patient has distinctive drusen and relevant family history, then the step before vision loss outbreak is categorized as AMD.

Macular degeneration is there is in occasional at the age more early.These situations many are caused by transgenation.Wherein have the hereditary macular digeneration of various ways, often kind of form all has its oneself clinical manifestation and genetic cause.The juvenile macular degeneration of known most common form is Stargardt disease, and it is with the heredity of autosomal recessive form.Patient was diagnosed usually below 20 years old.Although the progress of vision loss is different, these patients are to being exactly blind legally when 50 years old for major part.In ABCR gene, identify the sudden change causing Stargardt disease, described genes encoding transports the protein of retinoid through photoreceptor membrane.

Sight sensor progenitor cell of the present invention treatment degenerative disease in there is purposes, and can as progenitor cell, as its differentiation offspring (rod photoreceptor cell and cone cell) send, such as, after being orientated target photosensory cell system.Can to allow Transplanted cells or migrate to the retina site (such as there is stratum nucleare outside) of expection and to reconstruct or the mode in region of regeneration functional defect gives described cell.

In addition, the progenitor cell of genetic modification or sight sensor can also be used for site that gene product target is degenerated.These gene products can comprise promote survival the factor thus save natural degenerating neurons, can work in the mode of autocrine thus promote Cell survival and be divided into site-specific neurone or send neurotransmitter thus make functional rehabilitation) the factor.Ex vivo gene therapy (progenitor cell in such as modified recombinant culture or sight sensor) can use effectively as neuroprotective strategies; thus pass through somatomedin and neurotrophic factor (such as FGF2; NGF; Ciliary neurotrophic factor (CNTF); with Brain Derived Neurotrophic Factor (BDNF), the display of these nutritional factor can be slowed down the process of necrocytosis in retinal degeneration model significantly) and prevent in RP, AMD and glaucoma and cause the retina cell in the disease of retinal detachment to damage.Use the treatment of sight sensor progenitor cell and/or photoreceptor cell (can the combination of the synthetically grown factor or somatomedin through transformation) that the continual delivery of neuroprotective can not only be guaranteed, but also can the retina that damages of rebuild.

In the method for the invention, cell to be transplanted is transferred in receptor in the acceptable vehicle of any physiology, and wherein said vehicle is included in the isotonic excipient for preparing under giving the fully aseptic condition of the mankind.With regard to the General Principle of medical formulation, reader can with reference to CellTherapy:StemCellTransplantation, GeneTherapy, andCellularImmunotherapy, byG.Morstyn & W.Sheridaneds, CambridgeUniversityPress, 1996.The cellular excipient of composition and any adjoint element adjust according to the approach of administration and device.Described cell can by introducings such as injection, conduits.At frozen cell under liquid nitrogen temperature and standing storage, can use when thawing.If freezing, then cell is stored in 10%DMSO usually, in 50%FCS, 40%RPMI1640 substratum.

Pharmaceutical preparations of the present invention is optionally packaged in suitable container, and has the printed instructions for required object.This type of formulation can comprise the mixture of retina differentiation and/or the nutritional factor being applicable to form sight sensor progenitor cell or photoreceptor cell combined.Such composition can comprise further and is applicable to be transferred to the suitable buffer reagent in animal and/or vehicle.Such composition can comprise cell to be transplanted further.

Pharmaceutical preparations

Medicine acceptable carrier can be used to prepare PRPC or photoreceptor cell.Such as PRPC or photoreceptor cell can give separately, or give as the composition of pharmaceutical formulation.Any mode easily that the compound that topic is stated can be mixed with for medicinal use gives.The pharmaceutical formulation being applicable to give can comprise PRPC or photoreceptor cell, and combine the acceptable sterile isotonic of one or more medicines or non-isotonic solution (such as balanced salt solution (BSS)), dispersion liquid, suspension, emulsion or sterilized powder (this powder can reconstruct sterile injectable solution or dispersion liquid before use, and it can comprise antioxidant, buffer reagent, fungistat, solute or suspension or thickening material).Exemplary pharmaceutical preparations comprises PRPC or photoreceptor cell, and combines bSS (balanced salt solution, this solution of every mL comprises sodium-chlor 7.14mg, Repone K 0.38mg, calcium chloride dihydrate 0.154mg, magnesium chloride hexahydrate 0.2mg, Sodium phosphate dibasic 0.42mg, sodium bicarbonate 2.1mg, dextran 10 .92mg, glutathione bisulphide (Sleep-promoting factor B) 0.184mg, hydrochloric acid and/or sodium hydroxide (by pH regulator to about 7.4), Yu Shuizhong).

When giving the pharmaceutical preparations used in the disclosure, it can be the acceptable form of apyrogenic physiology.

The prepared product comprising PRPC or photoreceptor cell used in method as herein described can be transplanted with the form of suspension, gel, colloid, slurry or mixture.In addition, prepared product can be encapsulated ideally with viscous form or be injected in vitreous humour, for delivery to the site that retina or choroid damage.In addition, when injecting, the PRPC photoreceptor cell of cryopreservation can be resuspended in commercially available balanced salt solution, thus obtains the osmotic pressure needed for being given by subretinal injection and concentration.Prepared product can give not completely lose the surrounding's macular region in disease, and this can promote attachment and/or the survival of given cell.

PRPC and/or photoreceptor cell can freezing (cryopreservations) as described herein.When thawing, the vigor of this type of cell can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or about 100% (after such as thawing results cell at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or about 100% is great-hearted, or after thawing, at least 20% of initial frozen cell number, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or about 100% with vigor state results).

PRPC of the present disclosure or photoreceptor cell can be sent with the form of the acceptable ophthalmology formulation of medicine by intraocular injection.Such as when giving formulation by intravitreal injection, described solution can be concentrated, making it possible to send minimum volume.Concentration for injecting can for effective and nontoxic any amount, and this depends on the factor as herein described.Can with at least about 10 for the PRPC of patient treatment or the pharmaceutical preparations of photoreceptor cell ⁴the dosage formulation of individual cell/mL.Can with at least about 10 for the PRPC of patient treatment or photoreceptor cell prepared product ³, 10 ⁴, 10 ⁵, 10 ⁶, 107,10 ⁸, 10 ⁹, 10 ¹⁰the dosage formulation of individual PRPC or photoreceptor cell/mL.Such as, PRPC or photoreceptor cell can be prepared with the form of medicine acceptable carrier or vehicle.

The pharmaceutical formulation of PRPC as herein described or photoreceptor cell can comprise at least about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000 PRPCS or photoreceptor cell.The pharmaceutical formulation of PRPC or photoreceptor cell can comprise at least approximately 1x10 ⁴, 2x10 ⁴, 3x10 ⁴, 4x10 ⁴, 5x10 ⁴, 6x10 ⁴, 7x10 ⁴, 8x10 ⁴, 9x10 ⁴, 1x10 ⁵, 2x10 ^5,3x10 ⁵, 4x10 ⁵, 5x10 ⁵, 6x10 ⁵, 7x10 ⁵, 8x10 ⁵, 9x10 ⁵, 1x10 ⁶, 2x10 ⁶, 3x10 ⁶, 4x10 ⁶, 5x10 ⁶, 6x10 ⁶, 7x10 ⁶, 8x10 ⁶, 9x10 ⁶, 1x10 ⁷, 2x10 ⁷, 3x10 ⁷, 4x10 ⁷, 5x10 ⁷, 6x10 ⁷, 7x10 ⁷, 8x10 ⁷, 9x10 ⁷, 1x10 ⁸, 2x10 ⁸, 3x10 ⁸, 4x10 ⁸, 5x10 ⁸, 6x10 ⁸, 7x10 ⁸, 8x10 ⁸, 9x10 ⁸, 1x10 ⁹, 2x10 ⁹, 3x10 ⁹, 4x10 ⁹, 5x10 ⁹, 6x10 ⁹, 7x10 ⁹, 8x10 ⁹, 9x10 ⁹, 1x10 ¹⁰, 2x10 ¹⁰, 3x10 ¹⁰, 4x10 ¹⁰, 5x10 ¹⁰, 6x10 ¹⁰, 7x10 ¹⁰, 8x10 ¹⁰, 9x10 ¹⁰individual PRPC or photoreceptor cell.The pharmaceutical formulation of PRPC or photoreceptor cell can comprise at least approximately 1x10 ²-1x10 ³, 1x10 ²-1x10 ⁴, 1x10 ⁴-1x10 ⁵, or 1x10 ³-1x10 ⁶individual PRPC or PRPC or photoreceptor cell.The pharmaceutical formulation of PRPC or photoreceptor cell can comprise at least about 10,000,20,000,25,000,50,000,75,000,100,000,125,000,150,000,175,000,180,000,185,000,190,000 or 200,000 PRPC or photoreceptor cell.Such as, the pharmaceutical formulation of PRPC or photoreceptor cell can comprise at least about 20,000-200,000 PRPC or photoreceptor cell in the volume of at least about 50-200 μ L.In addition, the pharmaceutical formulation of PRPC or photoreceptor cell can comprise about 50 in the volume of 150 μ L, 000 PRPC or sight sensor, about 200 can be comprised in the volume of 150 μ L, 000 PRPC or photoreceptor cell, at least about 180 are comprised, 000 PRPC or photoreceptor cell in the volume of 150 μ L.

In aforesaid pharmaceutical preparations and composition, can by counting vigor cell get rid of the quantity that non-vigor cell measures PRPC or photoreceptor cell, or the concentration of PRPC or photoreceptor cell.Such as can by dyestuff alive (such as trypan blue) can not be got rid of or using function test (being such as attached to the ability of culture substrate, phagolysis etc.) detects PRPC or the sight sensor of non-vigor.In addition, one or more marks of the cell and/or the cell type of eliminating expression instruction except PRPC or sight sensor that can express the mark of one or more PRPC or photoreceptor cell by counting measure the quantity of PRPC or photoreceptor cell or the concentration of PRPC or photoreceptor cell.

PRPC or photoreceptor cell can be mixed with for being sent in the acceptable ophthalmology vehicle of medicine, prepared product and ocular surface are kept in touch to be enough to make the affected region of Premeabilisation of cells eyes (such as anterior chamber, back room, vitreum, aqueous humour, vitreous humour, cornea, iris/eyelash, crystalline, choroid, retina, sclera, on choroid space (suprachoridalspace), conjunctiva, subconjunctival space, episcleral space, cornea inner width, anterior corneal surface space (epicornealspace), annulus ciliaris (parsplana), to perform the operation the avascular area that causes or macula lutea) time.

PRPC or photoreceptor cell can be included in cellular layer.Such as can by substrate, (complete cellular layer can discharge by it, such as thermosensitive polymer, such as thermo-sensitivity NIPA (PNIPAAm) grafted surface, on a surface, cell can adhere to and breed under culture temperature, then when temperature variation, surface characteristic changes, cell cultures layer is caused to discharge (such as by being cooled to lower than lower Kraft point, LCST)) upper cultivate PRPC or photoreceptor cell prepare comprise PRPC or photoreceptor cell cellular layer (see aSilvaetal., TrendsBiotechnol.2007Dec, 25 (12): 577-83, Hsiueetal., Transplantation.2006Feb15, 81 (3): 473-6, Ide, T.etal. (2006), Biomaterials27,607-614, Sumide, T.etal. (2005), FASEBJ.20,392-394, Nishida, K.etal. (2004), Transplantation77,379-385, andNishida, K.etal. (2004), N.Engl.J.Med.351,1187-1196, every portion of these documents is all incorporated to herein in full with way of reference).Cellular layer can be attached to be applicable to transplant substrate on, the substrate such as can dissolved in vivo when cellular layer migrates in HOST ORGANISMS, such as can by being applicable to culturing cell in the substrate of transplanting or preparing by being released into by the cell obtained by another substrate (such as thermosensitive polymer) in the substrate that is applicable to transplant.The exemplary substrate being applicable to potentially transplant can comprise gelatin (see Hsiueetal. etc.).The alternative substrate going for transplanting comprises the matrix etc. based on fibrinogen.Cellular layer may be used for the medicine for the preparation of prevention or treatment retinal degenerative disease.PRPC or photoreceptor cell layer can be prepared in the eyes for being introduced into the study subject that this needs.Such as can transplant PRPC or photoreceptor cell layer by membranectomy under nest (subfovealmembranectomy) and cellular layer is introduced in eyes in need, or may be used for the medicine for the preparation of transplanting after membranectomy under nest.

The volume of the prepared product given according to method as herein described can depend on many factors, the type of the such as quantity of mode of administration, PRPC or photoreceptor cell, the age of patient and body weight and disease to be treated and seriousness.If given by injection, then the volume of the pharmaceutical preparations of PRPC of the present disclosure or photoreceptor cell can be at least about 1,1.5,2,2.5,3,4 or 5mL.Described volume can be at least about 1-2mL.If such as given by injection, then the volume of the pharmaceutical preparations of PRPC of the present disclosure or photoreceptor cell can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 μ L (microlitre).Such as, the volume of prepared product of the present disclosure can be at least about 10-50,20-50,25-50 or 1-200 μ L.The volume of prepared product of the present disclosure can be at least about 10,20,30,40,50,100,110,120,130,140,150,160,170,180,190,200 μ L or higher.

Such as, prepared product can comprise at least approximately 1x10 ³, 2x10 ³, 3x10 ³, 4x10 ³, 5x10 ³, 6x10 ³, 7x10 ³, 8x10 ³, 9x10 ³, 1x10 ⁴, 2x10 ⁴, 3x10 ⁴, 4x10 ⁴, 5x10 ⁴, 6x10 ⁴, 7x10 ⁴, 8x10 ⁴, 9x10 ⁴pRPC or photoreceptor cell/μ L.Prepared product can comprise 2000 PRPC or photoreceptor cell/μ L, and such as 100,000 PRPC or photoreceptor cell/50 μ L, or 180,000 PRPC or photoreceptor cell/90 μ L.

The method for the treatment of retinal degeneration may further include and gives immunosuppressor.Operable immunosuppressor includes but not limited to antilymphocyte globulin (ALG) (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azepine sulphur purine, (anti-IL-2R α receptor antibody), S-Neoral (cyclosporin A), (anti-IL-2R α receptor antibody), everolimus, mycophenolic acid, (anti-CD20 antibodies), sirolimus and tacrolimus.The dosage of immunosuppressor can be at least about 1,2,4,5,6,7,8,9 or 10mg/kg.When using immunosuppressor, they can system or local give, and they can before giving PRPC or photoreceptor cell, simultaneously or give afterwards.Immunosuppressant therapy can continue a few week, some months, several years indefinitely after giving PRPC or photoreceptor cell.Such as after giving PRPC or photoreceptor cell to patient, 5mg/kg S-Neoral can be given and reach 6 weeks.

The methods for the treatment of of retinal degeneration can comprise the PRPC or photoreceptor cell that give single dose.In addition, methods for the treatment of as herein described can comprise therapeutic process, wherein within for some time, repeatedly gives PRPC or photoreceptor cell.Exemplary therapeutic process can comprise weekly, every two weeks, monthly, quarterly, every half a year or treat once a year.Alternatively, treatment can be carried out within several period, gives multiple dosage (such as first week, dosage once a day) thus at first, needs less and more infrequently dosage subsequently.

If given by intraocular injection, then in the whole life-span of patient, periodically can send PRPC or photoreceptor cell by one or many.Such as can once a year, every 6-12 month once, every 3-6 month once, within every 1-3 month, once or often 1-4 week once transmit PRPC or photoreceptor cell.Alternatively, for some situation or disorder, it can be desirable for frequently giving.If given by transplant or device, then according to particular patient to be treated and needs that are disorderly or situation, in the whole life-span of patient, once or periodically one or many gives PRPC or photoreceptor cell.This type of ground, considers the treatment plan that can change within for some time.Such as can need at the beginning frequently to treat (such as every day or once in a week treatment).Through after a period of time, when the situation of patient is improved, the treatment of lower frequency can be needed or even do not need further treatment.

Method as herein described may further include carrys out the step of the effect of monitor therapy or prevention by measuring electroretinogram ERG reaction, acuity vision threshold value or luminance threshold in study subject.In addition, described method can also comprise carrys out the effect of monitor therapy or prevention by the immunogenicity of cell or the migration of cell in monitoring eyes.

PRPC or PR may be used for the medicine preparing treatment retinal degeneration.In addition, the disclosure covers the purposes of prepared product in treatment is blind comprising PRPC or PR.Such as, the prepared product comprising the mankind PRPC or PR may be used for treatment and changes the relevant retinal degeneration of disease with multiple vision, wherein said vision changes disease can cause photoreceptor damage and blind, such as diabetic retinopathy, macular degeneration (comprises age relevant macular degeneration, the macular degeneration that the such as Wet Age macular degeneration of being correlated with is relevant with dry age), retinitis pigmentosa, Stargardt disease (fundus flavimaculatus Stargardt disease), yctalopia and colour blindness.At least about 5 can be comprised in prepared product, 000-500,000 PRPC or PR (such as 10,000 PRPC or PR), its can be applied to retina with treatment with cause photosensory cell to damage and blind a series of visions change the retinal degeneration of disease-related, such as diabetic retinopathy, macular degeneration (comprise age relevant macular degeneration), retinitis pigmentosa and Stargardt disease (fundus flavimaculatus Stargardt disease).

PRPC or PR provided herein can be PRPC or PR.But note, human cell may be used in human patients, and in animal model or animal patient.Such as can check human cell in the mouse of retinal degeneration, rat, cat, dog or non-human primate model.In addition, human cell can be used for the treatment of animal in need to therapeutic, such as, in Veterinary Medicine.The example of beasts study subject or patient includes but not limited to dog, cat and other companion animals, and has the animal (such as domestic animal and horse) of economic worth.

Be below for illustration of embodiments of the invention, should do not regard it as and limit scope of the present invention.

Embodiment

embodiment 1:the generation of sight sensor progenitor cell

At Matrigel ^tM(the Solubilised preparations obtained by Engelbreth-Holm-Swarm (EHS) mice sarcoma cell, BDBiosciences) on, in mTESR1 substratum (StemCellTechnology), cultivator embryonic stem cell-like under without the condition of feeder layer.When 80-90% converges, make passage or freezing.Enzyme (Dispase) or non-enzymatic (based on the cell dispersal buffer reagent of EDTA, Invitrogen) technology is used to carry out going down to posterity of stem cell.

Direct differentiation method may be used for generating ocular progenitor cell, retina neural progenitor cell, sight sensor progenitor cell and retinal light injury photoreceptor.Without the need to forming embryoid body.

Group method in these embodiments for sight sensor research and development is schematically shown in Figure 19, and it further illustrates the substratum used in each step of described process.

According to dyeing data, be measured to cell and within 30 days, become EFPC (indicated by the dyeing implemented during at the 20th day at the 7th day-, which demonstrate the identity of this cell), they become RNPC (being indicated by the dyeing implemented during at the 30th day) for 45 days at the 21st day-, and they became PhRPC (dyeing based on implementing at the 90th day) at 1-4 month.

In addition, the method described in estimated service life embodiment 1, occurs that the opportunity of different cell type is as follows:

Ocular progenitor cell (EFPC): 7-30 days/purity is 65%-98%

Retina neural progenitor cell (RNPC): 21-45 days/purity is 70%-95%

Rod photoreceptor cell sight sensor and the sight sensor progenitor cell (PhRP) both cone cell sight sensor: 1-4 month can be become/purity is 85%-95%

Think the sight sensor progenitor cell (PhRP) losing or reduce the ability (but do not lose or reduce the ability becoming cone cell sight sensor) becoming rod photoreceptor cell sight sensor: 5-12 month/purity is 85%-95%.

0th day: when 15-20% converges, the cytodifferentiation of induction human pluripotent stem cell.Substratum is replaced with retina induction (RI) substratum: in RI substratum, add the DMEM/F12:450mg/mlD-glucose supplementing following composition, 100 units/ml penicillin, 100 μ g/ml Streptomycin sulphates, 1% (or optionally 0.1 to 5%) N2 supplement (Invitrogen), 0.2% (or optionally 0.05-2.0%) B27 supplement, 0.1mMMEM nonessential amino acid solution, 25 μ g/ml (or optionally 5-50 μ g/ml) human insulin.In addition, also comprise Smad inhibitor noggin, and when comprise concentration be 10-100ng/ml or be preferably 50ng/ml time, it increases the expression of ocular transcription factor.As shown in Figure 1, comprise the different factors and (comprise 50ng/ml noggin, 5ng/mlDkk1,5ng/mlIGF-1, or 5ng/ml noggin, the combination of 5ng/mlDkk1 and 5ng/mlIGF-1) expression level of ocular transcription factor in the ocular progenitor cell of differentiation can be affected at the 21st day.In these conditions, the expression that 50ng/ml noggin significantly can induce ocular progenitor cell marker thing is comprised.

RI culture media composition comprises following material:

N2:1% (1mlN2/100ml substratum)

B27：0.2％(0.2mlb27/100ml)

Human insulin: 20 μ g/ml (except the 5 μ g/ml Regular Insulin that N2 feeds).The ultimate density of Regular Insulin is 25 μ g/ml.

Noggin: ultimate density is 50ng/ml.

1st day-4 days: the replacing carrying out perfect medium every day.Although this frequency is preferred, think, if use the substratum of comparatively large vol, then more infrequently (such as every 2-3 days) replaced medium can be specially suitable.Under the concentration identical with above-mentioned steps, cell colony continued propagation in the RI substratum with Regular Insulin and noggin.Being exposed to RI substratum after 1 day, being positioned at the cell elongation of margin of colonies and forming cylindricality, as shown in Figure 2 A.

5th day: at the 5th day, cell culture reached 80-90% and converges.Substratum is replaced with Neural Differentiation (ND) substratum: the Neurobasal substratum (composition listed in Figure 21, Invitrogen), wherein supplement 450mg/mlD-glucose, 100 units/ml penicillin, 100 μ g/ml Streptomycin sulphates, 1 × GlutaMAX ^tM(by L-glutaminate, the dipeptides of the stabilized form that Ala-Gln obtains), 1% (or optionally 0.1 to 5%) N2 supplement (serum-free supplement that Chemical Composition is determined, it is based on Bottenstein ' sN-1 formulation, it comprises 1mM mankind's Transferrins,iron complexes (Holo), 0.0861mM Regular Insulin recombinant full-lenght chain, 0.002 progesterone, 10.01mM putrescine and 0.00301mM selenite, Invitrogen), 2% (or optionally 0.05-2.0%) B27 supplement (composition listed in figure 21), 0.1mMMEM nonessential amino acid solution.In addition, be added to by noggin in ND substratum, final concentration is 50ng/ml (or optionally 10-100ng/ml).

6th day-20 days: cell is remained in ND substratum.Within every 2 days, change the substratum of half amount.Cell colony continued propagation in ND substratum.Border cell becomes flat and large, and centrocyte is less and form the cell cluster (Fig. 2 B) of consolidation.At about 14th day, the cell being positioned at colony center started to form Rose spline structure (Fig. 2 C).At the 21st day, shown by immunostaining and flow cytometry, cell coexpression PAX6 and RX1 (Fig. 3 A-3B) more than 90%.By immunostaining, cell is nidogen and the SOX2 positive (Fig. 3 C-3D).It is negative that cell is ES cell sign thing (being specially OCT4) and retina neural progenitor cell marker thing (being specially CHX10).By RT-PCR, cell expressing ocular transcription factor: PAX6, RX1, LHX2, SIX3, SIX6, TBX3 and SOX2 (Fig. 3 E).These results show that cell is ocular progenitor cell.

After use Neural Differentiation culture medium culturing cell, from about 7-8 days (cultivating about 2-3 days ND substratum), cell became ocular progenitor cell.When these time points, there is the two positive cell of detectable pax6/rx1.After about 14th day (14-30 days), generate the ocular progenitor cell of high purity (>90%).

At about 7-30 days, formed " ocular progenitor cell " or " EFPC ".

21st day-24 days: at the 21st day, on the ND substratum without noggin, cell by outstanding on growth surface, and mechanically divides cluster.Cell cluster is transferred on the culture dish of the extremely low attachment of 100mm.In suspension culture, cell cluster is gathered and is formed single ball (solid bunch).At the 23rd day, replace the substratum of half.

At the 25th day, collect ball and remove dead cell and fragment by using the ball described in the washing of ND substratum.Cell ball is inoculated in coating Matrigel ^tMchamber slides (for immunostaining) or tissue culture dishes ND substratum in.Ball adhered in 12 hours.Their continued propagation also show neuronal phenotypes, particularly in ball, show cell aggregation, and wherein said ball extends the spinous process of aixs cylinder sample, and some cells are by moving out in aggregate (Fig. 4 A).Wherein have less large epithelioid cell, it eliminates in passage (" the 2nd month-3 months " in vide infra).Keep culture, within every 2 days, change the substratum of half, converge until cell culture becomes.The multiple balls observing described cell ball are attached on flat board.

At the 30th day, migrating cell was that Tuj1 is positive, and it indicates immature and ripe neurone (Fig. 4 B).Cell in aggregate is Tuj1 feminine gender.Cell (comprising in aggregate or by the cell moving out in aggregate) coexpression PAX6 and CHX10 more than 95%, shows that they form retina neural progenitor cell (Fig. 4 C).

2nd month-3 months: cell grows and goes down to posterity in ND substratum.When the cells reached confluency, the passage obtained by above-mentioned steps is made.2 step continuous passage technology are used for producing highly purified neural culture by eliminating most non-neuron phenotype cells.First step: neural ball is cultivated.Enzyme process (such as using Accutase) or mechanical means is adopted by cell to make cell dissociation become the mixture of individual cells and cell cluster.Cell is transferred in the ND substratum of low attachment culture dish.The all cells with neuronal phenotypes all forms neural ball in suspension culture.At the 3rd day, change the substratum of half, and keep cell until the 5th day.Second step: attachment is cultivated.Collect neural ball at the 5th day, and remove dead cell or fragment by using the ball described in the washing of ND substratum.Described ball is inoculated in coating Matrigel ^tMtissue culture dishes on, until converge.First step and second step hocket, and cell are so kept until 3rd month terminates.

At the end of 3rd month, described cell display neural phenotypes.Specifically, described cell forms spinous process (Fig. 5 A) in culture.They can be bred.As by immunostaining assessed, they express PAX6, but are CHX10 feminine gender (Fig. 5 B).By immunostaining, described cell is that recoverin is positive, and it expresses (Fig. 5 C) in the tenuigenin of cell paste.In addition, described cell also expresses Visual purple, opsin and recoverin mRNA (Fig. 5 D).Real-time PCR analysis shows that the expression of the transcription factor controlling rod photoreceptor cell and/or cone cell photoreceptor differentiation highly expresses (table 1).These results show that described cell is sight sensor progenitor cell.In addition, point at this moment, in the culture that makes discovery from observation, all or nearly all cell is sight sensor progenitor cell.

Transcription factor	Rod photoreceptor cell/cone cell	Change multiple (compared with ESC)
			TRβ2	Cone cell	3.5-5
NR2E3	Rod photoreceptor cell	7-11
			NRL	Rod photoreceptor cell	4-8
MASH1	Rod photoreceptor cell	1000-1200
			CRX	Rod photoreceptor cell, cone cell	-
RORβ	Rod photoreceptor cell, cone cell	40-60
			OTX2	Rod photoreceptor cell, cone cell	-

Table 1. controls the quantitative RT PCR analysis of the transcription factor of photoreceptor differentiation and regeneration

4th month-9 months/or longer: the ex vivo of sight sensor progenitor cell.In some embodiments, use 2 step continuous passage technology mentioned above to further expand cell (" the 2nd month-3 months ").But observe, through after a period of time, cell loses the ability (although they maintain the ability being divided into rod photoreceptor cell sight sensor) being divided into cone cell sight sensor.Specifically, by 2 step continuous passage technology sight sensor progenitor cell kept in culture 9 months, then differentiation-inducing after, they only produce the cell of expressing rod photoreceptor cell sight sensor mark, and do not produce the cell of expressing cone cell sight sensor mark.This character can potentially for favourable purposes, because the progenitor cell preferably producing rod photoreceptor cell sight sensor may be used for treating in the disease wherein needing to form rod photoreceptor cell, or be used for measuring in relevant research with sight sensor progenitor cell destiny as reagent.

embodiment 2:the differentiation of sight sensor progenitor cell: use vitamin A acid and taurine to carry out cell process

Under the following conditions; use sight sensor progenitor cell 2 weeks: the ND substratum of retinoic acid treatments attachment, wherein supplement 100ng/ml (or optionally 10-1000ng/ml) vitamin A acid and 100 μMs (or optionally 20-500 μM) taurine.Within every 2 days, change the substratum of half.

Cytodifferentiation is made: described substratum is replaced with photoreceptor differentiation substratum in photoreceptor differentiation substratum, it comprises Neurobasal substratum (Invitrogen), wherein supplement 450mg/mlD-glucose, 100 units/ml penicillin, 100 μ g/ml Streptomycin sulphates, 1 × GlutaMAX ^tM1%N2 supplement (Invitrogen), 2%B27 supplement (formula number 080085-SA), and add 5 μMs of (or optionally 1-100 μM) forskolins, 10ng/ml (or optionally 1-100ng/ml) BDNF, 10ng/ml (or optionally 1-100ng/ml) CNTF, 10ng/ml (or optionally 5-50ng/ml) LIF and 10 μM of (or optionally 1-100 μM) DATP.Within every 2 days, change the substratum of half.Specifically, the amount of each factor is as follows: forskolin (5 μMs), BDNF (10ng/ml), CNTF (10ng/ml), LIF (10ng/ml) and DATP (10 μMs).LIF is optional after measured, and can omit.

2 weeks after starting cytodifferentiation, Visual purple is detected in the tenuigenin and spinous process of cell paste, opsin (green/red), the expression (Fig. 6 A-6D) of recoverin and phosphodiesterase 6A α subunit (PDE6a).The expression of results of these genes shows that they are photoreceptor cells.

embodiment 3:the cryopreservation of the retina neural progenitor cell that mankind ESC derives

The sight sensor progenitor cell of retina neural progenitor cell of the present invention, sight sensor progenitor cell of the present invention and retinoic acid treatments of the present invention can be frozen in non-animal derived cryopreservation buffer reagent (such as CryostorCS10) or another kind of cryopreservation buffer reagent (such as 90%FBS and 10%DMSO).For sight sensor progenitor cell, observe and become neural ball to be favourable cell freezing, this may be the benefit due to cell and cells contacting.Preferably, neural ball is freezing with not too large size, such as 50-250 cell.

embodiment 4:zooscopy is carried out in the animal model of Stargardt macular dystrophy

Zooscopy is carried out in the animal model (ELOVL4 transgenosis 2 (TG2) mouse (Fig. 7)) of Stargardt macular dystrophy.

Use Accutase; by sight sensor progenitor cell (producing as described in example 1 above); and use the sight sensor progenitor cell (that is, immature photoreceptor cell, produces as described in example 2 above) of vitamin A acid and taurine process to be separated into single cell.Cell is suspended in PBS buffer reagent again.

28 the largest TG2 mouse accept 1 μ l cell suspension at subretinal space and (comprise 5 × 10 ⁵individual cell) injection, or accept 150 μ l cell suspensions at caudal vein and (comprise 1 × 10 ⁶individual cell) injection.All mouse all experienced baseline ERG and OCT and check before injection cell.

The water that use supplements cyclosporin A (USP modification) raises mouse.

When injecting 1 month after cell, the obvious improvement of the mouse display rod photoreceptor cell sight sensor function of subretinal injection sight sensor progenitor cell, this is shown (Fig. 8) by the remarkable increase of the twilight vision ERG amplitude of a ripple and b ripple.Tail vein injection sight sensor progenitor cell and obviously improving through the mouse display rod photoreceptor cell sight sensor function of the sight sensor progenitor cell of vitamin A acid and taurine process, this is shown (Fig. 9) by the remarkable increase of the twilight vision ERG amplitude of a ripple and b ripple.

When injecting 2 months after cell, tail vein injection improves further through the mouse display rod photoreceptor cell sight sensor function of the sight sensor progenitor cell of retinoic acid treatments, and this is shown (Figure 10 A-10C) by a ripple of twilight vision ERG response curve and the further increase of b wave amplitude.The function of cone cell sight sensor is significantly improved, and this is shown (Figure 11) by the enlarging markedly of vision improvement ERG amplitude of a ripple and b ripple.

During after injection 2 months, the mouse of tail vein injection prematurity photoreceptor cell (use vitamin A acid and taurine process) shows overall retinal thickness (being shown by OCT) significantly increases (Figure 12).

During after Transplanted cells 2 months, in the mouse of sight sensor progenitor cell accepting vitamin A acid and taurine process, in retina ONL, sight sensor neurone has obvious reservation (Figure 13).

Embodiment 5: monochromatism (colour blindness) and improve the animal model of scotopic vision

The cell that method according to embodiment 1 or embodiment 2 produces is checked in the mouse of monochromatism (colour blindness), sheep and/or dog model.Use with drag:

Mouse: (1) cpfl5 mouse: the mouse model with the achroous natural formation of CNGA3 sudden change; (2) CNGA3 knock-out mice; (3) GNAT2cpfl3 mouse: the sudden change relevant with GNAT2; (4) PDE6C-cpfl1: the sudden change relevant with pde6c.

Sheep: Awassi sheep lamb: the sudden change in CNGA3

Dog: the Canis animals having identified the 2 kinds of natural formation suddenlyd change in CNGA3: show the way in dog Alaska husky and German undercoat, autosomal recessive Canis animals cone cell degenerates.

Use Accutase that sight sensor progenitor cell (producing as described in example 1 above) is separated into single cell.Cell is suspended in PBS buffer reagent again.Injection 2 × 10 is accepted in the vitreous space of animal ⁵individual cell or more, or accept injection 5 × 10 in tail vein (such as caudal vein) ⁶individual cell or more.Control animal accepts injection PBS buffer reagent.Behind 1 or 2 months or the time point at other, give optomotor response inspection, to check sight function, to detect its possible improvement to animal.In addition, implement histologic analysis thus measure sight sensor neurone whether to have and significantly to retain or whether sight sensor neurone has obvious growth, in addition, detect the cell be transplanted in vitreous space whether to show good survival and cell after injection and whether be divided into the rod photoreceptor cell or cone cell photoreceptor cell of expressing its mark.

embodiment 6:zooscopy in sight sensor degeneration rat model (RoyalCollegeofSurgeons (RCS) rat)

Use Accutase, make sight sensor progenitor cell (producing as described in example 1 above) be separated into single cell.Cell is suspended in PBS buffer reagent again.

The 30th day after birth, to the intravitreal 2 × 10 of RCS rat ⁵individual cell, or to tail vein injection 5 × 10 ⁶individual cell.Control rats accepts the injection of PBS buffer reagent.

The water that use supplements cyclosporin A (USP modification) raises mouse.

When injecting 1 month after cell and 2 months, giving optomotor response to rat and checking, to check sight function.In treated rat, sight function does not improve significantly (data are not shown).

Can be checked by optomotor response, ERG, luminance threshold record and/or Optic center blood flow test detect the impact that sight function is produced.

2 months after injection cell, histology showed that, in the RCS rat giving cell therapy, sight sensor neurone obviously retains (Figure 15) in amphiblestroid ONL.

Being retained in cell therapy group of the rod photoreceptor cell shown by the immunostaining of Visual purple (rod photoreceptor cell) and opsin (cone cell) and cone cell sight sensor outer sections is all observed (in vitreum and tail vein injection, Figure 16 and Figure 17).

Migrate to cell in vitreous space after injection 2 months time show good survival, be then divided into the rod photoreceptor cell photoreceptor cell (Figure 18) of expressing rod photoreceptor cell sight sensor mark further.

Claims

1. a substantially pure prepared product for sight sensor progenitor cell, it comprises:

Multiple sight sensor progenitor cell; And

Be applicable to the medium of the vigor of the sight sensor progenitor cell described in keeping,

Wherein detected by qPCR, the described cell more than 90% in described prepared product is PAX6+ and CHX10-in immunocytochemistry, and mRNA transcript is the MASH1 positive.

2. a prepared product for sight sensor progenitor cell, it comprises:

Multiple cell, it comprises the sight sensor progenitor cell of at least 50%; And

Be applicable to the medium of the vigor keeping described sight sensor progenitor cell,

Wherein detected by qPCR, described sight sensor progenitor cell is PAX6 (+) and CHX10 (-) in immunocytochemistry, and mRNA transcript is the MASH1 positive.

3. a prepared product for sight sensor progenitor cell, it comprises:

Multiple sight sensor progenitor cell, it is not substantially containing multipotential stem cell, retinal ganglial cells, ripe sight sensor and/or amacrine cell; And

4. be applicable to a pharmaceutical preparations for the sight sensor progenitor cell of mammalian subject, it comprises:

A () multiple sight sensor progenitor cell, is wherein detected by qPCR, the described cell more than 90% in described prepared product is PAX6+ and CHX10-in immunocytochemistry, and mRNA transcript is the MASH1 positive; And

B () is for keeping described sight sensor progenitor cell transplantation to the medicine acceptable carrier of the vigor in mammalian subject.

5. one kind comprises at least 10 ⁹the low temperature cellular preparations of individual sight sensor progenitor cell (PRPC), it comprises:

B cryopreservation system that () is compatible with the vigor of described sight sensor progenitor cell when thawing.

6. the prepared product in claim 1-5 described in any one, wherein said sight sensor progenitor cell is derived from multipotential stem cell.

7. prepared product according to claim 6, wherein said multipotential stem cell is selected from the multipotential stem cell of hESC and induction.

8. the prepared product in the claims described in any one, wherein said sight sensor progenitor cell is human cell.

9. the prepared product in the claims described in any one, is wherein detected by qPCR, and the sight sensor progenitor cell described in major part is the mRNA transcript positive for Nr2e3, Tr β 2, ROR β and NRO.

10. the prepared product in the claims described in any one, wherein said sight sensor progenitor cell is for retina neural progenitor cell, express one or more marks of more than at least 2 times, this mark is selected from: uPA, tenascin-C, CXCL16, CX3CL1 and chitinase 3 sample albumen 1, as by secretory protein is carried out immunoassay or by qPCR test mRNA transcript level detect.

Prepared product in 11. the claims described in any one, wherein in cell culture, described sight sensor progenitor cell has the replication of experience at least 20 population doublings, and during to described the 20th time multiplication, be less than the described cell generation necrocytosis of 25%, old and feeble or be divided into the cell of non-sight sensor in phenotype.

Prepared product in 12. the claims described in any one, the Transferrins,iron complexes of wherein said sight sensor progenitor cell and/or Transferrins,iron complexes mRNA level in-site are than glyceraldehyde 3 phosphate dehydrogenase protein or mRNA level in-site respectively low at least 25%.

Prepared product in 13. the claims described in any one, wherein said sight sensor progenitor cell is that HLA genotype is consistent.

Prepared product in 14. the claims described in any one, wherein said sight sensor progenitor cell is that genome is consistent.

Prepared product in 15. the claims described in any one, wherein said sight sensor progenitor cell has the mean terminal Restriction Fragment Length (TRF) being longer than 8kb.

Prepared product in 16. the claims described in any one, wherein derivative relative to fetus sight sensor, the content and/or the enzymic activity that participate in following one or more protein in described sight sensor progenitor cell have statistically reduction significantly: the adjustment of (i) cell cycle and cell senescence; (ii) cellular energy and/or lipid metabolism; (iii) apoptosis.

Prepared product in 17. the claims described in any one, for wherein derivative relative to fetus sight sensor, participate in the content of cytoskeletal structure and the cytokinetic protein relevant with it and/or enzymic activity in described sight sensor progenitor cell and there is statistically significant rising there is statistical significance.

18. prepared products according to claim 4, it is applicable to give human patients.

19. prepared products according to claim 4, it is applicable to give non-human beasts patient.

Prepared product described in 20. claims 6 or 7, wherein said sight sensor progenitor cell is originated by common multipotential stem cell to break up.

21. prepared products according to claim 1, are wherein applicable to keep the described medium of the vigor of described sight sensor progenitor cell to be selected from developing medium, cryopreservation agent and be applicable to the biocompatibility injectable media of human patients injection.

Prepared product in 22. the claims described in any one, wherein said sight sensor progenitor cell keeps the plasticity-being divided into both rod photoreceptor cell and cone cell.

Prepared product in 23. the claims described in any one, wherein when the transplanted subretinal space to ELOVL4-TG2 mouse, described sight sensor progenitor cell migrates to outside in ELOVL4-TG2 mouse stratum nucleare, and improves twilight vision and vision improvement ERG reaction.

24. pharmaceutical preparations according to claim 4, wherein said prepared product is apyrogenic and without fungi.

Prepared product in 25. the claims described in any one, wherein said sight sensor progenitor cell has activate the phagocytic capacity, optionally engulfs sight sensor outer sections, the pHrodo of separation ^tMredE.coliBioParticles or the ability of the two.

Prepared product in 26. the claims described in any one, wherein said sight sensor progenitor cell secretes one or more neuroprotective factor.

Substantially the pure prepared product of the photoreceptor cell that 27. 1 kinds of multipotential stem cells derive, it comprises:

A photoreceptor cell that () multipotential stem cell is derivative, the described cell wherein more than 90% is PAX6+, CHX10-in immunocytochemistry, and be Visual purple+and/or opsin+; And

B () is applicable to the medium of the vigor of the stem cell-derived photoreceptor cell described in maintenance.

28. pharmaceutical preparations being applicable to the sight sensor of mammalian subject, it comprises:

(b) medicine acceptable carrier for keeping described photoreceptor cell to migrate to the vigor in mammalian subject.

29. 1 kinds of methods for the treatment of disease or the disorder caused by photoreceptor loss in patient, it comprise give the pharmaceutical preparations of sight sensor progenitor cell according to claim 4 or the pharmaceutical preparations of photoreceptor cell according to claim 28 or the two.

30. methods according to claim 29, wherein said cellular preparations is injected in the subretinal space of described patient.

31. 1 kinds of methods producing sight sensor progenitor cell, it comprises the following steps:

The for some time being enough to form individual cells ball is cultivated under the culture condition that ocular progenitor cell is replaced between low attachment condition and non-attachment condition, under being then in attachment condition,

Wherein continue alternately to change culture condition, until most described cell is sight sensor progenitor cell, wherein said sight sensor progenitor cell is characterized by PAX6 (+) and CHX10 (-), and wherein said sight sensor progenitor cell is divided into photoreceptor cell when using retinoic acid treatments.

32. 1 kinds of methods producing sight sensor progenitor cell, it comprises the following steps:

A ocular progenitor cell is cultivated by () in Neural Differentiation substratum is enough to make cell cluster form for some time of individual cells ball, wherein said ocular progenitor cell is preferably cell cluster, and preferably under the condition of low attachment or non-attachment, wherein said ocular progenitor cell is characterized by PAX6 (+), RX1 (+), OCT4 (-) and NANOG (-), and be preferably also characterized by SIX3 (+), SIX6 (+), LHX2 (+), TBX3 (+), SOX2 (+) and nidogen+, as passed through immunostaining and/or Flow Cytometry Assay,

B () is under attachment condition, preferably on biomaterial scaffolds, described cell ball is cultivated in Neural Differentiation substratum, until most cell is characterized by PAX6 (+) in described culture, the retina neural progenitor cell of CHX10 (+) and SOX2 (-), wherein said biomaterial scaffolds is such as gelatin, alginate, collagen protein 1 type, Matrigel ^tM, PGA, collagen protein, fibrinogen or self-assembling peptides;

C () after this, between low attachment and the condition of non-attachment, one or many alternately changes culture condition, continue for some time that described retina neural progenitor cell is enough to be formed individual cells ball, then under attachment condition, cultivate the cell ball comprising retina neural progenitor cell, wherein continue alternately to change culture condition until most cell is sight sensor progenitor cell, wherein said sight sensor progenitor cell is characterized by PAX6 (+) and CHX10 (-), and be preferably also characterized by Mash1, Nr2e3, Tr β 2, the mRNA transcript of ROR β and NRO is positive, as qPCR detect, and wherein said sight sensor progenitor cell is divided into photoreceptor cell when using retinoic acid treatments.

Method in 33. claim 29-32 described in any one, wherein said sight sensor progenitor cell is derived by multipotential stem cell.

34. methods according to claim 33, wherein said multipotential stem cell is the multipotential stem cell of embryonic stem cell or induction, is optionally the multipotential stem cell of hESC or mankind's induction.

Method described in 35. claims 33 or 34, wherein said sight sensor progenitor cell is not substantially to provide containing the form of multipotential stem cell.

Method in 36. claim 29-35 described in any one, wherein said sight sensor progenitor cell is at least 50%, at least 75% relative to other cell type, at least 85%, at least 95%, at least 99% or about 100% pure.

Method in 37. claim 29-36 described in any one, it comprises the further step of sight sensor progenitor cell described in cryopreservation.

The method of the substantially pure culture of 38. 1 kinds of sight sensor progenitor cells derived for the preparation of multipotential stem cell, it comprises:

A () cultivates multipotential stem cell in without the system of raising, thus produce one or more ocular progenitor cells;

One or more ocular progenitor cells described in (b) cultivation, thus produce the retina neural progenitor cell of PAX6+ and CHX10+;

Retina neural progenitor cell described in (c) cultivation, thus produce the sight sensor progenitor cell (PRPC) of PAX6+ and CHX10-.